Guobo Shen

Quantitative protein expression profiling of 14-3-3 isoforms in human renal carcinoma shows 14-3-3 epsilon is involved in limitedly increasing renal cell proliferation.

14‐3‐3 proteins regulate many cellular processes that are implicated in cancer development, and the seven 14‐3‐3 isoforms have different expression level and isoform‐specific roles in different tumors. However, the biological functions of 14‐3‐3 proteins and their correlations with renal carcinoma have not been investigated so far. In our study, the expression profiles and functional characterization of 14‐3‐3 proteins were discovered by a sensitive stable isotope labeling with amino acids in cell culture based quantitative proteomics analysis in human renal carcinoma tissues. We found that 14‐3‐3ε was up‐regulated with 1.44‐fold changes in renal cancerous tissues compared with that in counterpart kidney tissues, and 14‐3‐3σ was almost not detected in both tissues due to its DNA highly methylated in our previous reports. The other five isoforms almost have similar expression level in two states of renal tissues. The following RT‐PCR, Western blot and immunohistochemistry analysis for specific 14‐3‐3 isoform expression were all consistent with the quantitative proteomic data. Furthermore, the overexpression of 14‐3‐3ε in vitro can limitedly prompt the abnormal growth of renal tumor cells.

Inhibition of FGFR signaling by PD173074 improves antitumor immunity and impairs breast cancer metastasis.

Aberrant fibroblast growth factor (FGF) and FGF receptor (FGFR) system have been associated with breast cancer. The objectives of our study were to investigate the effects and mechanisms of FGFR inhibition on tumor growth and metastasis on breast cancer. Our studies showed that the FGFR inhibitor PD173074 decreased the viability of several human breast cancer cells, as well as 4T1 murine mammary tumor cells. Therefore, we chose 4T1 cells to study PD173074’s antitumor mechanism. Flow cytometry showed that PD173074 induced 4T1 cell apoptosis in a concentration-dependent manner. Western blot demonstrated that PD173074-induced apoptosis was correlated with the inhibition of Mcl-1 and survivin. Moreover, PD173074 also significantly increased the ratio of Bax/Bcl-2. PD173074 could also block 4T1 cell migration and invasion in vitro. In 4T1 tumor-bearing mice, PD173074 significantly inhibited tumor growth without obvious side effects. Meanwhile, PD173074 functionally reduced microvessel density and proliferation index and induced tumor apoptosis. Importantly, we found that FGFR inhibition by PD173074 reduced myeloid-derived suppressor cells (MDSCs) in the blood, spleens and tumors, accompanied by the increased infiltration of CD4+ and CD8+ T cells in the spleens and tumors. Furthermore, PD173074 significantly inhibited breast tumor metastasis to the lung of inoculated 4T1 breast cancer cells, which was accompanied by a reduction in MDSCs. Our findings suggested that FGFR inhibition could delay breast tumor progression, impair lung metastasis and break immunosuppression by effecting on tumor microenvironment, which may provide a promising therapeutic approach for breast cancer patient.

Isoform-specific expression and characterization of 14-3-3 proteins in human glioma tissues discovered by stable isotope labeling with amino acids in cell culture-based proteomic analysis.

Human 14‐3‐3 proteins have isoform‐specific expression and functions in different kinds of normal or tumor cells and tissues. However, the expression profiling of 14‐3‐3 proteins and isoform‐specific biological functions are unclear in human glioma so far. In our study, the expression levels and characterization of 14‐3‐3 isoforms in human glioma tissues were investigated by a sensitive, accurate stable isotope labeling with amino acids in cell culture‐based quantitative proteomic strategy. As a result, except unexpressed 14‐3‐3σ, the other six isoforms, with different expression levels, were existed in glioma tissues and para‐cancerous brain tissues (PBTs). 14‐3‐3β and η were upregulated, whereas 14‐3‐3ζ was downregulated in glioma tissues compared with that in PBTs. And the other three isoforms 14‐3‐3ε, θ, and γ had similar expression levels in human glioma tissues and PBTs. Western blot and immunohistochemistry analysis were both consistent with the quantitative proteomic data. The loss of expression of 14‐3‐3σ was further discovered due to DNA high methylation in its coding region in glioma by methylation‐specific PCR analysis. These results indicated that the four isoforms, including 14‐3‐3β, η, ζ, and σ, may play important roles in tumorigenesis of human glioma, which is probably used as potential biomarkers for diagnosis and targets for treatment of human gliomas in future.

Comparisons of three polyethyleneimine-derived nanoparticles as a gene therapy delivery system for renal cell carcinoma

BackgroundPolyethyleneimine (PEI), which can interact with negatively charged DNA through electrostatic interaction to form nanocomplexes, has been widely attempted to use as a gene delivery system. However, PEI has some defects that are not fit for keeping on gene expression. Therefore, some modifications against PEI properties have been done to improve their application value in gene delivery. In this study, three modified PEI derivatives, including poly(ε-caprolactone)-pluronic-poly(ε-caprolactone) grafted PEI (PCFC-g-PEI), folic acid-PCFC-isophorone diidocyanate-PEI (FA-PEAs) and heparin-PEI (HPEI), were evaluated in terms of their cytotoxicity and transfection efficiency in vitro and in vivo in order to ascertain their potential application in gene therapy.MethodsMTT assay and a marker GFP gene, encoding green fluorescent protein, were used to evaluate cell toxicity and transfection activity of the three modified PEI in vitro. Renal cell carcinoma (RCC) models were established in BALB/c nude mice inoculated with OS-RC-2 cells to detect the gene therapy effects using the three PEI-derived nanoparticles as gene delivery vehicles. The expression status of a target gene Von Hippel-Lindau (VHL) in treated tumor tissues was analyzed by semiquantitative RT-PCR and immunohistochemistry.ResultsEach of three modified PEI-derived biomaterials had an increased transfection efficiency and a lower cytotoxicity compared with its precursor PEI with 25-kD or 2-kD molecule weight in vitro. And the mean tumor volume was obviously decreased 30% by using FA-PEAs to transfer VHL plasmids to treat mice RCC models. The VHL gene expression was greatly improved in the VHL-treated group. While there was no obvious tumor inhibition treated by PCFC-g-PEI:VHL and HPEI:VHL complexes.ConclusionsThe three modified PEI-derived biomaterials, including PCFC-g-PEI, FA-PEAs and HPEI, had an increased transfection efficiency in vitro and obviously lower toxicities compared with their precursor PEI molecules. The FA-PEAs probably provide a potential gene delivery system to treat RCC even other cancers in future.

Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts☆

Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p<0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy.

Cathepsin B as a potential prognostic and therapeutic marker for human lung squamous cell carcinoma

BackgroundThe lung squamous cell carcinoma survival rate is very poor despite multimodal treatment. It is urgent to discover novel candidate biomarkers for prognostic assessment and therapeutic targets to lung squamous cell carcinoma (SCC).ResultsHerein a two-dimensional gel electrophoresis and ESI-Q-TOF MS/MS-based proteomic approach was used to identify differentially expressed proteins between lung SCC and adjacent normal tissues. 31 proteins with significant alteration were identified. These proteins were mainly involved in metabolism, calcium ion binding, signal transduction and so on. Cathepsin B (CTSB) was one of the most significantly altered proteins and was confirmed by western blotting. Immunohistochemistry showed the correlation between higher CTSB expression and lower survival rate. No statistically significant difference between CTSB-shRNA treated group and the controls was observed in tumor volume, tumor weight, proliferation and apoptosis. However, the CTSB-shRNA significantly inhibited tumor metastases and prolonged survival in LL/2 metastatic model. Moreover, CTSB, Shh and Ptch were up-regulated in patients with metastatic lung SCC, suggesting that hedgehog signaling might be activated in metastatic lung SCC which could affect the expression of CTSB that influence the invasive activity of lung SCC.ConclusionsThese data suggested that CTSB might serve as a prognostic and therapeutic marker for lung SCC.

Gene expression and methylation status of 14‐3‐3σ in human renal carcinoma tissues

Loss of 14‐3‐3σ expression mainly by methylation‐mediated silencing has been reported in several human cancers, but the methylation status of 14‐3‐3σ in human renal carcinoma is rarely studied so far. In this report, 14‐3‐3σ expression was first examined by RT‐PCR and immunohistochemistry, and further we investigated the methylation status by methylation‐specific PCR and the correlation between 14‐3‐3σ expression and its methylation. We found 14‐3‐3σ expression was lost in 27 of 31 renal tissues including 16 renal carcinoma tissues, eight para‐cancerous kidney tissues and seven normal kidney tissues. Among 16 renal carcinoma tissues, 14 cases had complete hypermethylation of 14‐3‐3σ. Eight para‐cancerous kidney tissues were almost completely methylated except one case had both methylation and unmethylation. Among seven normal kidney tissues, five cases had partial methylation, and the other two cases were completely methylated. In addition, 14‐3‐3σ mRNA had weak expression in OS‐RC‐2 cells, but it increased with gradual demethylation after treatment by a demethylation agent, 5‐aza‐2′‐deoxycytidine. In general, 14‐3‐3σ mRNA was mostly unexpressed, and its DNA frequently hypermethylated within 14‐3‐3σ coding region was closely associated with the gene silencing in cancerous and para‐cancerous kidney tissues. 14‐3‐3σ was also frequently methylated and almost silencing in normal kidney tissues. However, the methylation frequency was gradually reinforced with the extent of malignancy from normal to para‐cancerous and cancerous kidney tissues.

Dopamine receptor antagonist thioridazine inhibits tumor growth in a murine breast cancer model

Neuropsychological factors have been shown to influence tumor progression and therapeutic response. The present study investigated the effect of the dopamine receptor antagonist thioridazine on murine breast cancer. The anti-tumor efficacy of thioridazine was assessed using a murine breast cancer model. Cell apoptosis and proliferation were analyzed in vitro using flow cytometry (FCM) and the MTT assay, respectively. Western blot analysis was performed to assess Akt, phosphorylated (p)-Akt, signal transducer and activator of transcription (STAT) 3, p-STAT3 and p-p65 in tumor cells following treatment with thioridazine. The Ki67 index and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells were assessed in the tumor sections. Thioridazine was found to reduce tumor growth, inhibit tumor cell proliferation and induce apoptosis in a dose- and time-dependent manner in vitro. Thioridazine was also found to markedly inhibit tumor proliferation and induce tumor cell apoptosis in vivo as shown by the lower Ki67 index and increase in TUNEL-positive cells. In addition, thioridazine was observed to inhibit the activation of the canonical nuclear factor κ-light-chain-enhancer of activated B cells pathway and exert anti-tumor effects by remodeling the tumor stroma, as well as inhibit angiogenesis in the tumor microenvironment. In conclusion, thioridazine was found to significantly inhibit breast tumor growth and the potential for thioridazine to be used in cancer therapy may be re-evaluated and investigated in clinical settings.

Antitumoral efficacy by systemic delivery of heparin conjugated polyethylenimine-plasmid interleukin-15 complexes in murine models of lung metastasis.

Gene therapy shows promising application in cancer therapy, but the lack of an ideal gene delivery system is still a tough challenge for cancer gene therapy. Previously, we prepared a novel cationic nanogel, heparin‐polyethylenimine (HPEI), which had potential application in gene delivery. In the present study, we constructed a plasmid with high expression efficiency of interleukin‐15 (IL15) and investigated the effects HPEI–plasmid IL15 (HPEI–pIL15) complexes on the distribution level of the lung. We then evaluated the anticancer effect of HPEI–pIL15 complexes on lung metastases of B16‐F10 melanoma and CT26 colon carcinoma. These results demonstrated that intravenous injection of the HPEI–pIL15 complex exhibited the highest plasmid distribution level in the lung compared with that of PEI2K–pIL15 and PEI25K–pIL15, and mice treated with HPEI–pIL15 had a lower tumor metastasis index compared with other treatment groups. Moreover, the number of natural killer cells, which were intermingled among the tumor cells, and the level of tumor necrosis factor‐α and interferon‐γ in the serum also increased in the pIL15‐treated mice. Furthermore, the cytotoxic activity of spleen cells also increased significantly in the HPEI–pIL15 group. In addition, induction of apoptosis and inhibition of cell proliferation in lung tumor foci in the HPEI–pIL15 group was observed. Taken together, treating lung metastasis cancer with the HPEI nanogels delivered by plasmid IL15 might be a new and interesting cancer gene therapy protocol. (Cancer Sci 2011; 102: 1403–1409)

Downregulated expression of HSP27 in human low-grade glioma tissues discovered by a quantitative proteomic analysis

BackgroundHeat shock proteins (HSPs), including mainly HSP110, HSP90, HSP70, HSP60 and small HSP families, are evolutionary conserved proteins involved in various cellular processes. Abnormal expression of HSPs has been detected in several tumor types, which indicates that specific HSPs have different prognostic significance for different tumors. In the current studies, the expression profiling of HSPs in human low-grade glioma tissues (HGTs) were investigated using a sensitive, accurate SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative proteomic strategy.ResultsThe five HSP family members were detected and quantified in both HGTs and autologous para-cancerous brain tissues (PBTs) by the SILAC-based mass spectrometry (MS) simultaneously. HSP90 AB1, HSP A5(70 KDa), and especially HSP27 were significantly downregulated in HGTs, whereas the expression level of HSPA9 (70 KDa) was little higher in HGTs than that in PBTs. It was noted that the downregulation ratio of HSP27 was 0.48-fold in HGTs versus PBTs, which was further validated by results from RT-PCR, western blotting and immunohistochemistry. Furthermore, we detected HSP27 expression changes along with cell growth under heat shock treatment in glioma H4 cells.ConclusionThe SILAC-MS technique is an applicable and efficient novel method, with a high-throughput manner, to quantitatively compare the relative expression level of HSPs in brain tumors. Different HSP family members have specific protein expression levels in human low-grade glioma discovered by SILAC-MS analysis. HSP27 expression was obviously downregulated in HGTs versus PBTs, and it exhibited temporal and spatial variation under heat shock treatment (43°C/0-3 h) in vitro. HSP27s rapid upregulation was probably correlated with the temporary resistance to heat shock in order to maintain the survival of human glioma cells.