[Show abstract][Hide abstract] ABSTRACT: The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS) for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs), which are abundant in solid tumors, can be utilized for identification of rearranged ends.
PLoS ONE 01/2014; 9(6):e100089. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRT's limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes.
PLoS ONE 01/2013; 8(7):e69414. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microtubule inhibitors (MTIs) such as Taxol have been used for treating various malignant tumors. Although MTIs have been known to induce cell death through mitotic arrest, other mechanisms can operate in MTI-induced cell death. Especially, the role of p53 in this process has been controversial for a long time. Here we investigated the function of p53 in Taxol-induced apoptosis using p53 wild type and p53 null cancer cell lines. p53 was upregulated upon Taxol treatment in p53 wild type cells and deletion of p53 diminished Taxol-induced apoptosis. p53 target proteins including MDM2, p21, BAX, and β-isoform of PUMA were also upregulated by Taxol in p53 wild type cells. Conversely, when the wild type p53 was re-introduced into two different p53 null cancer cell lines, Taxol-induced apoptosis was enhanced. Among post-translational modifications that affect p53 stability and function, p53 acetylation, rather than phosphorylation, increased significantly in Taxol-treated cells. When acetylation was enhanced by anti-Sirt1 siRNA or an HDAC inhibitor, Taxol-induced apoptosis was enhanced, which was not observed in p53 null cells. When an acetylation-defective mutant of p53 was re-introduced to p53 null cells, apoptosis was partially reduced compared to the re-introduction of the wild type p53. Thus, p53 plays a pro-apoptotic role in Taxol-induced apoptosis and acetylation of p53 contributes to this pro-apoptotic function in response to Taxol in several human cancer cell lines, suggesting that enhancing acetylation of p53 could have potential implication for increasing the sensitivity of cancer cells to Taxol.
[Show abstract][Hide abstract] ABSTRACT: Proliferation activity has long been known to be one of the strongest prognostic factors in many different cancers. Nevertheless, microscopic evaluation of mitotic figures remains time-consuming and, furthermore, is relatively subjective. As the expression of cytoskeleton-associated protein 2 (CKAP2) is closely related to the mitotic phase, CKAP2 was evaluated as a surrogate mitotic figure (MF) marker.
A monoclonal antibody specific to human CKAP2 was produced, and immunohistochemistry was performed on normal tissue array sections and 30 breast cancer tissues.
The expression of CKAP2 in the normal human tissues was limited to well-known cell proliferation zones. Strong, readily visible, condensed chromatin staining of CKAP2 was observed specifically in mitotic cells, and the number of these cells was tightly correlated with the MF count in breast cancer tissues (P < 0.001, ρ = 0.743), suggesting its usefulness as a surrogate marker for MF counting.
Immunohistochemical staining with CKAP2 monoclonal antibody can be considered to be a new, effective approach to the assessment of proliferation activity in cancer tissues.
Journal of Cancer Research and Clinical Oncology 01/2012; 138(1):95-102. · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expression of transglutaminase 2 (TGase 2) is related to invasion and resistance to chemotherapeutic agents in several cancer cells. However, there has been only limited clinical validation of TGase 2 as an independent prognostic marker in cancer.
The significance of TGase 2 expression as an invasive/migratory factor was addressed by in vitro assays employing down-regulation of TGase 2. TGase 2 expression as a prognostic indicator was assessed in 429 Korean patients with early-stage non-small cell lung cancer (NSCLC) by immunohistochemical staining.
TGase 2 expression increased the invasive and migratory properties of NSCLC cells in vitro, which might be related to the induction of MMP-9. In the analysis of the immunohistochemical staining, TGase 2 expression in tumors was significantly correlated with recurrence in NSCLC (p = 0.005) or in the non-adenocarcinoma subtype (p = 0.031). Additionally, a multivariate analysis also showed a significant correlation between strong TGase 2 expression and shorter disease-free survival (DFS) in NSCLC (p = 0.029 and HR = 1.554) and in the non-adenocarcinoma subtype (p = 0.030 and HR = 2.184). However, the correlation in the adenocarcinoma subtype was not significant.
TGase 2 expression was significantly correlated with recurrence and shorter DFS in NSCLC, especially in the non-adenocarcinoma subtype including squamous cell carcinoma.
Molecular Cancer 09/2011; 10:119. · 5.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell therapy using MSCs (mesenchymal stem cells) might be effective treatment for refractory GVHD (graft-versus-host disease). However, the fate and distribution of MSCs after transplantation remains unclear. In this study, an animal model was developed to monitor the dynamic distribution of MSCs in mice with GVHD. A GVHD mouse model was established by transplanting C57BL/6 donor bone marrow cells and C57BL/6 EGFP (enhanced green fluorescent protein) splenocytes into lethally irradiated BALB/c nude recipient mice. Donor MSCs were obtained from MHC-identical C57BL/6 RFP (red fluorescent protein) mice and infused into the recipient mice on the same transplantation day. In vivo movement of the donor splenocytes (EGFP) and MSCs (RFP) were evaluated by measuring the biofluorescence (IVIS-Xenogen system). Donor splenocytes and MSCs reached the lungs first, and then the gastrointestinal tract, lymph nodes and skin, in that order; the transit time and localization site of these cells were very similar. In the recipient mouse with GVHD, the number of detectable cells declined with time, as assessed by biofluorescence imaging and confirmed by RT (real-time)-PCR. This bioimaging system might be useful for preclinical testing and the design of therapeutic strategies for monitoring the dynamic distribution of MSCs with GVHD.
Cell Biology International 11/2010; 35(4):417-21. · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Graft-versus-host disease (GvHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Recent literature demonstrates a potential benefit of human mesenchymal stromal cells (MSC) for the treatment of refractory GvHD; however, the optimal dose remains uncertain. We set out to develop an animal model that can be used to study the effect of MSC on GvHD.
A GvHD mouse model was established by transplanting C3H/he donor bone marrow (BM) cells and spleen cells into lethally irradiated BALB/c recipient mice. MSC were obtained from C3H/he mice and the C3H/10T1/2 murine MSC line.
The mRNA expression of Foxp3 in regional lymph nodes (LN) localized with T cells was markedly increased by the addition of C3H10T1/2 cells in a real-time polymerase chain reaction (PCR). Using a mixed lymphocyte reaction, we determined the optimal splenocyte proliferation inhibition dose (MSC:splenocyte ratios 1:2 and 1:1). Three different C3H10T1/2 cell doses (low, 0.5 x 10(6), intermediate, 1 x 10(6), and high, 2 x 10(6)) with a consistent splenocyte dose (1 x 10(6)) were evaluated for their therapeutic potential in an in vivo GvHD model. The clinical and histologic GvHD score and Kaplan-Meier survival rate were improved after MSC transplantation, and these results demonstrated a dose-dependent inhibition.
We conclude that MSC inhibit GvHD in a dose-dependent manner in this mouse model and this model can be used to study the effects of MSC on GvHD.
[Show abstract][Hide abstract] ABSTRACT: Recently, it was reported that expression of transglutaminase 2 plays an important role in doxorubicin/cisplatin resistance in breast and ovarian cancer. The aims of this study were to verify the role of transglutaminase 2 in cisplatin response in non-small cell lung cancer (NSCLC) and to study if transglutaminase 2 gene (TGM2) methylation can be a molecular marker for good response to cisplatin.
TGM2 promoter methylation was analyzed by sodium bisulfite sequencing. Cisplatin sensitivity was analyzed by treatment of cisplatin in NSCLC cell lines with/without TGM2 or TGM2 siRNA transfection.
In one-third of NSCLC cell lines, TGase 2 gene (TGM2) was silenced by promoter methylation. The TGM2 promoter-methylated cell lines (HCC-95 and HCC-1588) showed relatively higher sensitivity to cisplatin than the TGM2-expressing cell lines (NCI-H1299 and HCC-1195). Down-regulation and over-expression of TGM2 in those NSCLC cells also suggested a positive correlation of cisplatin sensitivity and TGM2 inhibition. With doxorubicin, the relationship was quite similar.
We showed that good responders of cisplatin in NSCLC could be identified by the promoter methylation of TGM2 and that TGase 2 inhibition appears to be an effective cisplatin-sensitizing modality in NSCLC.
Journal of Cancer Research and Clinical Oncology 09/2009; 136(4):493-502. · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inonotus obliquus (Chaga mushroom), one of the widely known medicinal mushrooms, has been used to treat various cancers in Russia and most of Baltic countries for many centuries.
To examine the anti-proliferative effects of Inonotus obliquus extract on melanoma B16-F10 cells. Furthermore, to assess the anti-tumor effect of Inonotus obliquus extract in vivo in Balb/c mice.
The water extract of Inonotus obliquus was studied for anti-proliferative effects on the growth and morphology of B16-F10 melanoma cells and for anti-tumor effect using in vivo in Balb/c mice.
Inonotus obliquus extract not only inhibited the growth of B16-F10 cells by causing cell cycle arrest at G(0)/G(1) phase and apoptosis, but also induced cell differentiation. These effects were associated with the down-regulation of pRb, p53 and p27 expression levels, and further showed that Inonotus obliquus extract resulted in a G(0)/G(1) cell cycle arrest with reduction of cyclin E/D1 and Cdk 2/4 expression levels. Furthermore, the anti-tumor effect of Inonotus obliquus extract was assessed in vivo in Balb/c mice. Intraperitoneal administration of Inonotus obliquus extract significantly inhibited the growth of tumor mass in B16-F10 cells implanted mice, resulting in a 3-fold (relative to the positive control, (*)p<0.05) inhibit at dose of 20mg/kg/day for 10 days.
This study showed that the water extract of Inonotus obliquus mushroom exhibited a potential anticancer activity against B16-F10 melanoma cells in vitro and in vivo through the inhibition of proliferation and induction of differentiation and apoptosis of cancer cells.
Journal of Ethnopharmacology 11/2008; 121(2):221-8. · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the anti-proliferative and apoptotic effects of Chaga mushroom (Inonotus obliquus) water extract on human hepatoma cell lines, HepG2 and Hep3B cells.
The cytotoxicity of Chaga extract was screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Morphological observation, flow cytometry analysis, Western blot were employed to elucidate the cytotoxic mechanism of Chaga extract.
HepG2 cells were more sensitive to Chaga extract than Hep3B cells, as demonstrated by markedly reduced cell viability. Chaga extract inhibited the cell growth in a dose-dependent manner, which was accompanied with G0/G1-phase arrest and apoptotic cell death. In addition, G0/G1 arrest in the cell cycle was closely associated with down-regulation of p53, pRb, p27, cyclins D1, D2, E, cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 expression.
Chaga mushroom may provide a new therapeutic option, as a potential anticancer agent, in the treatment of hepatoma.
World Journal of Gastroenterology 02/2008; 14(4):511-7. · 2.55 Impact Factor