• Asian Pacific Journal of Cancer Prevention
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• v.13 no.3
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• pp.821-825
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• 2012

Purpose: Centromere protein H (CENP-H) and Ki67 are overexpressed in some malignancies, but whether they are predictors of survival after primary resection for hypopharyngeal squamous cell carcinoma (HSCC) remains unknown. Methods: We assessed immunohistochemical expression of CENP-H and Ki67 in 112 HSCC specimens collected between March 2003 and March 2005 for analysis by clinical characteristics. The Kaplan-Meier method was used to analyze relapse-free survival and logistic multivariate regression to determine risk factors of relapse-free survival. Cholecystokinin octapeptide assays and flow cytometry were used to examine cell proliferation and apoptosis after siRNA inhibition of CENP-H in HSCC cells. Results: Overall, 50 (44.6%) HSCC specimens showed upregulated CENP-H expression and 69 (61.6%) upregulated Ki67. An increased CENP-H protein level was associated with advanced cancer stage and alcohol history (P=0.012 and P=0.048, respectively) but an increased Ki67 protein level only with advanced cancer stage (P=0.021). Increased CENP-H or Ki67 were associated with short relapse-free survival (P<0.001 or P=0.009, respectively) and were independent predictors of relapse-free survival (P=0.001 and P=0.018, respectively). siRNA knockdown of CENP-H mRNA inhibited cell proliferation and promoted cancer cell apoptosis in vitro. Conclusions: Upregulated CENP-H and Ki67 levels are significantly associated with short relapse-free survival in HSCC. These factors may be predictors of a relapsing phenotype in HSSC cases.

• Journal of Ginseng Research
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• v.46 no.3
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• pp.481-488
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• 2022

Background: Although the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet. The chromosomal instability resulting from dysregulated mitotic processes is usually increased in cancer. In this study, we aimed to investigate the anticancer effects of ginsenoside Rg1 on mitotic progression in cancer. Materials and methods: Cancer cells were treated with ginsenoside Rg1 and their morphology and intensity of different protein were analyzed using immunofluorescence microscopy. The level of proteins in chromosomes was compared through chromosomal fractionation and Western blot analyses. The location and intensity of proteins in the chromosome were confirmed through immunostaining of mitotic chromosome after spreading. The colony formation assays were conducted using various cancer cell lines. Results: Ginsenoside Rg1 reduced cancer cell proliferation in some cancers through inducing mitotic arrest. Mechanistically, it inhibits the phosphorylation of histone H3 Thr3 (H3T3ph) mediated by Haspin kinase and concomitant recruitment of chromosomal passenger complex (CPC) to the centromere. Depletion of Aurora B at the centromere led to abnormal centromere integrity and spindle dynamics, thereby causing mitotic defects, such as increase in the width of the metaphase plate and spindle instability, resulting in delayed mitotic progression and cancer cell proliferation. Conclusion: Ginsenoside Rg1 reduces the level of Aurora B at the centromere via perturbing Haspin kinase activity and concurrent H3T3ph. Therefore, ginsenoside Rg1 suppresses cancer cell proliferation through impeding mitotic processes, such as chromosome alignment and spindle dynamics, upon depletion of Aurora B from the centromere.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.912-918
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• 2003

Mitotic centromere-associated kinesin (MCAK), which is a novel kinesin with a central motor domain, is believed to playa role in mitotic segregation of chromosome during the M phase of the cell cycle. In the present study, it is shown that a rabbit polyclonal antibody has been produced using the N-terminal region (187 aa) of human MCAK expressed in E. coli as the antigen. To express the N-terminal region in E. coli, the MCAK cDNA fragment encoding N-terminal 187 aa was obtained by PCR and was then inserted into the pET 3d expression vector. Molecular mass of the N-terminal region overexpressed in the presence of IPTG was 23.2 kDa on SDS-PAGE, and the protein was insoluble and mainly localized in the inclusion body that could be easily purified from the other cellular proteins. The N-terminal region was purified by electro-elution from the gel after the inclusion body was resolved on the SDS-PAGE. The antiserum obtained after tertiary immunization with the purified protein specifically recognized HsMCAK when subjected to Western blot analysis, and showed a fluctuation of the protein level during the cell cycle of human Jurkat T cells. Synchronization of the cell-cycle progression required for recovery of cells at a specific stage of the cell cycle was performed by either hydroxyurea or nocadazole, and subsequent release from each blocking at 2, 4, and 7 h. Northern and Western analyses revealed that both mRNA and protein of HsMCAK reached a maximum level in the S phase and declined to a basal level in the G1 phase. These results indicate that a polyclonal antibody raised against the N-terminal region (187 aa) of HsMCAK, overexpressed in E. coli, specifically detects HsMCAK (81 kDa), and it can analyze the differential expression of HsMCAK protein during the cell cycle.

• Journal of Life Science
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• v.15 no.2 s.69
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• pp.253-260
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• 2005

Mitotic centromere-associated kinesin (MCAK), which is a member of the Kin I (internal motor domain) subfamily of kinesin-related proteins, is known to play a role in mitotic segregation of chromosome during M phase of the cell cycle. In the present study, we have produced a rat polyclonal antibody using human MCAK (HsMCAK) expressed in E. coli as the antigen. The antibody specifically recognized the HsMCAK protein (81 kDa), and could detect its nuclear localization in human Jurkat T cells and 293T cells by Western blot analysis. The specific stage of the cell cycle was obtained through blocking by either hydroxyl urea or nocodazole and subsequent releasing from each blocking for 2, 4, and 7 h. While the protein level of HsMCAK reached a maximum level in the S phase with slight decline in the $G_{2}-M$ phase, the electrophoretic mobility shift from $p81^{MCAK}\;to\;p84^{MCAK}$ began to be induced in the late S phase and reached a maximum level in the $G_{2}/M $ phase, and then it disappeared as the cells enter into the $G_{1}$ phase. Immunocytochemical analysis revealed that HsMCAK protein localized to centrosome and nucleus at the interphase, whereas it appeared to localize to the spindle pole, centromere of the condensed mitotic DNA, spindle fiber, or midbody, depending on the specific stage of the M phase. These results demonstrate that a rat polyclonal antibody raised against recombinant HsMCAK expressed in E. coli specifically detects human MCAK, and indicate that the electrophoretic mobility shift from $p81^{MCAK}\;to\;p84^{MCAK}$, which may be associated with its differential intracellular localization during the cell cycle, fluctuates with a maximum level of the shift at the $G_{2}-M$ phase.