Lantu Gou

Comparative Proteomics Approach to Screening of Potential Diagnostic and Therapeutic Targets for Oral Squamous Cell Carcinoma

This work demonstrates that a comprehensive strategy of proteomics identification combined with further validation and detailed functional analysis should be adopted in the field of cancer biomarker discovery. A comparative proteomics approach was utilized to identify differentially expressed proteins in 10 oral squamous carcinoma samples paired with their corresponding normal tissues. A total of 52 significantly and consistently altered proteins were identified with eight of these being reported for the first time in oral squamous carcinoma. Of the eight newly implicated proteins, RACK1 was chosen for detailed analysis. RACK1 was demonstrated to be up-regulated in cancer at both the mRNA and protein levels. Immunohistochemical examination showed that the enhanced expression of RACK1 was correlated with the severity of the epithelial dysplasia as well as clinical stage, lymph node involvement, and recurrence, which are known indicators of a relatively poor prognosis in oral squamous carcinoma patients. RNA interference specifically targeted to silence RACK1 could initiate apoptosis of oral squamous carcinoma cells. Taken together, the results indicate that RACK1 is up-regulated in oral squamous carcinoma, not only being closely related to cell proliferation and apoptosis but also linked to clinical invasiveness and metastasis in carcinogenesis. The observations suggest that RACK1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of oral squamous carcinoma. Further this comprehensive strategy could be used for identifying other differentially expressed proteins that have potential to be candidate biomarkers of oral squamous carcinoma.

Proteomic profiling identifies aberrant epigenetic modifications induced by hepatitis B virus X protein.

The hepatitis B virus-encoded X (HBx) protein coactivates transcription of a variety of viral and cellular genes and it is believed to play essential roles in viral replication and hepatocarcinogenesis. To examine the pleiotropic effects of HBx protein on host cell protein expression, we utilized 2-DE and MS analysis to compare and identify differentially expressed proteins between a stable HBx-transfected cell line (HepG2-HBx), constitutively expressing HBx, and vector control cells. Of the 60 spots identified as differentially expressed (+/- over 2-fold, p < 0.05) between the two cell lines, 54 spots were positively identified by MS/MS analysis. Several recent studies suggested that HBx was involved in regional hypermethylation of tumor suppressor genes and global hypomethylation of satellite 2 repeats during hepatocarcinogenesis; however, no specific gene has been reported as hypomethylated by HBx. Promoter methylation analysis was examined for those protein spots showing significant alterations, and our results revealed that specific genes, such as aldehyde dehydrogenase 1 (ALDH1), can be hypomethylated by HBx, and two calcium ion-binding proteins, S100A6 and S100A4, were hypermethylated by HBx and could be re-expressed by AZA (DNA methylase inhibitor) treatment. Moreover, via cluster and pathway analysis, we proposed a hypothetical model for the HBx regulatory circuit involving aberrant methylation of retinol metabolism-related genes and calcium homeostasis-related genes. In summary, we profiled proteome alterations between HepG2-HBx and control cells, and found that HBx not only induces regional hypermethylation but also specific hypomethylation of host cell genes.

Identification of ATP synthase beta subunit (ATPB) on the cell surface as a non-small cell lung cancer (NSCLC) associated antigen

BackgroundAntibody-based immuneotherapy has achieved some success for cancer. But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far. It is essential to identify more immunogenic antigens (especially cellular membrane markers) for tumor diagnosis and therapy.MethodsThe membrane proteins of lung adenocarcinoma cell line A549 were used to immunize the BALB/c mice. A monoclonal antibody 4E7 (McAb4E7) was produced with hybridoma technique. MTT cell proliferation assay was carried out to evaluate the inhibitory effect of McAb4E7 on A549 cells. Flow cytometric assay, immunohistochemistry, western blot and proteomic technologies based on 2-DE and mass spectrometry were employed to detect and identify the corresponding antigen of McAb4E7.ResultsThe monoclonal antibody 4E7 (McAb4E7) specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells. By the proteomic technologies, we identified that ATP synthase beta subunit (ATPB) was the corresponding antigen of McAb4E7. Then, flow cytometric analysis demonstrated the localization of the targeting antigen of McAb4E7 was on the A549 cells surface. Furthermore, immunohistochemstry showed that the antigen of McAb4E7 mainly aberrantly expressed in tumor cellular membrane in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC). The rate of ectopic expressed ATPB in the cellular membrane in lung adenocarcinoma, squamous carcinoma and their adjacent nontumourous lung tissues was 71.88%, 66.67% and 25.81% respectively.ConclusionIn the present study, we identified that the ectopic ATPB in tumor cellular membrane was the non-small cell lung cancer (NSCLC) associated antigen. ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

Proteomic analysis of interstitial fluid in bone marrow identified that peroxiredoxin 2 regulates H(2)O(2) level of bone marrow during aging.

Hematopoiesis in bone marrow declines during aging owing to alteration of the hematopoietic niche. However, due to difficult accessibility and other complexities, senescence-related alteration of the hematopoietic niche is largely unknown. The interstitial fluid of bone marrow (IFBM), a pivotal component of the hematopoietic niche, includes soluble secretory factors that are present between bone marrow cells. To characterize the proteomic profile changes of IFBM during aging, we analyzed the IFBMs of young, adult, and senescent rats using 2-DE combined with ESI/MALDI-Q-TOF MS. Finally, 31 differentially expressed proteins involved in multiple biological functions were identified. Peroxiredoxin 2 (Prx2), down-regulated during aging, was further analyzed and demonstrated that it is produced by bone marrow stromal cells. Interestingly, higher levels of hydrogen peroxide (H(2)O(2)) were detected in the bone marrow with lower Prx2 expression. Moreover, exogenous Prx2 reduced the intracellular H(2)O(2) level in bone marrow stromal cells in vitro. Therefore, Prx2 is implied in the regulation of H(2)O(2) production in the bone marrow during aging. Our data characterized the dynamic protein profiles of the bone marrow microenvironment during aging and we provided clues to elucidate the mechanism of creating a low ROS level in the hematopoietic niche.

Proteomic identification of RhoA as a potential biomarker for proliferation and metastasis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and there is an urgent need to discover novel factors that can act as biomarkers for prognostic assessment and therapeutic targets of HCC. In this study, highly purified plasma membrane proteins from clinical tissue samples were obtained using a strategy combining sucrose density gradient centrifugation and subsequent phase partition. Using a two-dimensional gel electrophoresis and MALDI-Q-TOF MS/MS-based proteomics approach, we identified 13 plasma membrane-associated proteins that were differentially expressed in HCC and normal liver tissues. Of those, RhoA was one of the most significantly upregulated proteins in HCC, and its overexpression was confirmed using Western blotting. Immunohistochemistry suggested a link between RhoA expression and poor differentiation and clinicopathologic stage. Suppression of RhoA expression in HepG2 and Hep3B cells by RNA interference led to significant inhibition of cell growth, induction of apoptosis, and a decrease in migration. Our data suggest that RhoA may serve as a potential biomarker and an attractive therapeutic target for HCC.

A novel pro-apoptosis gene PNAS4 that induces apoptosis in A549 human lung adenocarcinoma cells and inhibits tumor growth in mice

The gene PNAS4 is a high conservative gene that shares high homology of sequence in various organisms from plants to animals. We found overexpression of human PNAS4 induced apoptosis and arrested cell cycle in S phase in A549 human lung adenocarcinoma cells. In C57BL/6 mice model of Lewis lung carcinoma, overexpression of mouse PNAS4 significantly suppressed tumor growth and prolonged survival time through induction of tumor cell apoptosis, exhibiting effective antitumor. Our original investigations in vitro and vivo indicated PNAS4 is a novel pro-apoptosis gene, which could be used as a potential target of cancer biotherapy in future.

Isoliquiritigenin inhibits the growth of multiple myeloma via blocking IL-6 signaling.

Previous studies have suggested that isoliquiritigenin (ISL) has anti-carcinogenic activity in several kinds of solid tumors, however, little is known about the effects of ISL on hematologic malignancies. In this study, we investigated the effects of ISL on multiple myeloma (MM) cells both in vitro and in vivo. The results showed that ISL could inhibit the growth of MM cells and induce their apoptosis in time- and dose-dependent manners. ISL exhibited significant anti-tumor activity in MM xenograft models and synergistically enhanced the anti-myeloma activity of adriamycin. Further analysis demonstrated that ISL not only downregulated IL-6 expression but also significantly decreased levels of phosphorylated ERK and STAT3 and could inhibit phosphorylation levels of ERK and STAT3 induced by recombinant human IL-6, which are critical signaling proteins in IL-6 signaling regulation networks. Taken together, our findings suggested that ISL could inhibit the growth of MM via blocking IL-6 signaling and might serve as a promising therapeutic agent for treatment of MM.

Pnas4 is a novel regulator for convergence and extension during vertebrate gastrulation

Recent studies show that human Pnas4 might be tumor associated, while its function remains unknown. Here, we investigate the developmental function of Pnas4 using zebrafish as a model system. Knocking down Pnas4 causes gastrulation defects with a shorter and broader axis, as well as a posteriorly mis‐positioned prechordal plate, due to the defective convergence and extension movement. Conversely, over‐expression of Pnas4 mRNA leads to an elongated body axis. We further demonstrate that Pnas4 is required cell‐autonomously for dorsal convergence but not for anterior migration. In addition, genetic interaction assays indicate that Pnas4 might act in parallel with non‐canonical Wnt signal in the regulation of cell movement. Our data suggest that Pnas4 is a key regulator of cell movement during gastrulation.

Improved therapeutic efficacy against murine carcinoma by combining honokiol with gene therapy of PNAS-4, a novel pro-apoptotic gene.

PNAS‐4, a novel pro‐apoptotic gene activated during the early response to DNA damage, can inhibit proliferation via apoptosis when overexpressed in some tumor cells. Recent studies have indicated that honokiol can induce apoptosis, inhibit angiogenesis, and suppress tumor growth. In the present study, we investigated whether mouse PNAS‐4 (mPNAS‐4) could augment the apoptosis of tumor cells induced by honokiol in vitro, and whether the antiangiogenic activity of honokiol and induction of apoptosis by mPNAS‐4 could work cooperatively to improve the antitumor efficacy in vivo. In vitro, mPNAS‐4 inhibited proliferation of murine colorectal carcinoma CT26 and Lewis lung carcinoma LL2 cells through induction of apoptosis, and significantly augmented the apoptosis of CT26 and LL2 cells induced by honokiol. Compared with treatment with mPNAS‐4 or honokiol alone, in vivo systemic administration of an expression plasmid encoding mPNAS‐4 and low‐dose honokiol significantly suppressed tumor growth through the enhanced induction of apoptosis and the augmented inhibition of angiogenesis. Our data suggest that the combined treatment with mPNAS‐4 plus honokiol augments antitumor effects in vitro and in vivo, and that the improved antitumor activity in vivo may be associated with enhanced induction of apoptosis and augmented inhibition of angiogenesis. The present study may provide a novel and effective method for the treatment of cancer. (Cancer Sci 2009; 100: 1757–1766)

Eradication of growth of HER2-positive ovarian cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate in mouse xenograft model.

Objective Ovarian cancer is 1 kind of a highly malignant gynecologic tumor, and current treatments have not achieved satisfactory effects. Human epidermal growth factor receptor 2 (HER2)–targeted therapies including trastuzumab and trastuzumab-DM1 (T-DM1) (antibody-cytotoxic drug conjugates) have been applied to treat HER2-overexpressing breast cancers in clinic. In the present study, we explored whether T-DM1 could effectively treat HER2-positive human ovarian carcinoma in vitro and in vivo. Methods HER2 expressions of 6 ovarian cancer cell lines and 2 breast carcinoma cell lines were validated, and the binding capacity of T-DM1 to HER2-positive ovarian cancer SKOV3 cells were analyzed by flow cytometry. Nude mice bearing intraperitoneal and subcutaneous SKOV3 xenografts were used to investigate the antitumor effect of T-DM1. Results High HER2 expressions in SKOV3 cell lines were detected. The binding capacity of T-DM1 to HER2-positive SKOV3 cells was in a similar manner comparing with trastuzumab. In vitro, T-DM1 showed strong growth inhibitory on SKOV3 cells, with IC50 values of 0.15 nmol/L. Nude mice bearing intraperitoneal and subcutaneous SKOV3 xenografts were used to investigate the antitumor effects of T-DM1 in vivo. In subcutaneous xenografts model, T-DM1 (30 mg/kg and 10 mg/kg) indicated significant anticancer effects. It is noteworthy that tumors were completely eradicated in the T-DM1 (30 mg/kg) group, and no regrowth was observed in a long time after the termination of the treatment. In the peritoneal xenograft model, tumor nodules in 3 of 7 mice were hardly observed in the abdominal cavity of mice after intraperitoneal injection of T-DM1 (30 mg/kg). At the same time, tumor nodules from the other 4 mice weighed on the average of only 0.07 g versus 1.77 g in control group. Conclusions Our data showed that T-DM1 possessed promising antitumor effects on HER2-overexpressing ovarian cancer in mouse model, which provided valuable references for the future clinical trials.