• Journal of Life Science
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• v.27 no.8
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• pp.864-872
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• 2017

Equine chorionic gonadotropin (eCG) consists of highly glycosylated ${\alpha}-$ and ${\beta}-subunits$ and is a unique member of the gonadotropin family, because it elicits the response characteristics of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. To directly assess the biological function of $rec-eCG{\beta}/{\alpha}$, we constructed mammalian expressing vectors of equine luteinizing hormone/chorionic gonadotropin receptors (eLH/CGR). The activity of $rec-eCG{\beta}/{\alpha}$ in vitro assayed in transient transfected CHO-K1 cells and in stably transfected PathHunter Parental cells with eLH/CGR was investigated. $rec-eCG{\beta}/{\alpha}$ was efficiently secreted in the CHO-K1 suspension cell media, and the quantity detected was about 200 mIU/ml from 1 to 7 days after transfection. In the western blot analysis, the $rec-eCG{\beta}/{\alpha}$ protein was broadly identified to be about 40~45 kDa molecular weight. The cAMP stimulation in CHO-K1 cells expressing eLH/CGR was determined to evaluate the activity of $rec-eCG{\beta}/{\alpha}$. The cAMP concentration increased in direct proportion to the concentration of the $rec-eCG{\beta}/{\alpha}$. The $EC_{50}$ value in the transient transfected CHO-K1 cells was $8.1{\pm}6.5ng$. The stable cell lines of eLH/CGR were established in the PathHunter Parental cells expressing ${\beta}-arrestin$. We found that $rec-eCG{\beta}/{\alpha}$ had full LH activity in the PathHunter Parental cells expressing eLH/CGR. The $EC_{50}$ value in transient and stable cells was $5.0{\pm}4.7ng/ml$ and $4.5{\pm}5.2ng/ml$, respectively. These results suggest that $rec-eCG{\beta}/{\alpha}$ has a biological activity in a cell expressing eLH/CGR. These stable cells expressed in PathHunter Parental cells could be useful for elucidating the functional mechanisms of deglycosylated $rec-eCG{\beta}/{\alpha}$ mutants.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.6191-6196
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• 2012

Aims and Background: Traditional chemotherapy strategies for human leukemia commonly use drugs based on cytotoxicity to eradicate cancer cells. One predicament is that substantial damage to normal tissues is likely to occur in the course of standard treatments. Obviously, it is urgent to explore therapies that can effectively eliminate malignant cells without affecting normal cells. Our previous studies indicated that ginsenoside $Rg_1$ ($Rg_1$), a major active pharmacological ingredient of ginseng, could delay normal hematopoietic stem cell senescence. However, whether $Rg_1$ can induce cancer cell senescence is still unclear. Methods: In the current study, human leukemia K562 cells were subjected to $Rg_1$ exposure. The optimal drug concentration and duration with K562 cells was obtained by MTT colorimetric test. Effects of $Rg_1$ on cell cycle were analyzed using flow cytometry and by SA-${\beta}$-Gal staining. Colony-forming ability was measured by colony-assay. Telomere lengths were assessed by Southern blotting and expression of senescence-associated proteins P21, P16 and RB by Western blotting. Ultrastructural morphology changes were observed by transmission electron microscopy. Results: K562 cells demonstrated a maximum proliferation inhibition rate with an $Rg_1$ concentration of $20{\mu}\;mol{\cdot}L^{-1}$ for 48h, the cells exhibiting dramatic morphological alterations including an enlarged and flat cellular morphology, larger mitochondria and increased number of lysosomes. Senescence associated-${\beta}$-galactosidase (SA-${\beta}$-Gal) activity was increased. K562 cells also had decreased ability for colony formation, and shortened telomere length as well as reduction of proliferating potential and arrestin $G_2$/M phase after $Rg_1$ interaction. The senescence associated proteins P21, P16 and RB were significantly up-regulated. Conclusion: Ginsenoside $Rg_1$ can induce a state of senescence in human leukemia K562 cells, which is associated with p21-Rb and p16-Rb pathways.