Human Osteosarcoma Cell
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2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Ming-Yang Liu ◽  
Fei Liu ◽  
Yan-Jiao Li ◽  
Jia-Ning Yin ◽  
Yan-Li Gao ◽  
...  

The function and mechanism underlying the suppression of human osteosarcoma cells by ginsenoside-Rg5 (Rg5) was investigated in the present study. MG-63, HOS, and U2OS cell proliferation was determined by MTT assay after Rg5 treatment for 24 h. Rg5 inhibited human osteosarcoma cell proliferation effectively in a dose-dependent manner. The range of effective inhibitory concentrations was 160-1280 nM. Annexin V-FITC and PI double-staining assay revealed that Rg5 induced human osteosarcoma cell apoptosis. Western blotting, qRT-PCR, and FACS experiments revealed that Rg5 inhibited human osteosarcoma cells via caspase-3 activity which was related to the LC3-mediated autophagy pathway. Rg5 decreased the phosphorylation of PI3K, Akt, and mTORC1 activation. In contrast, LC3-mediated autophagy and caspase-3 activity increased significantly. A PI3K/AKT stimulator, IGF-1, reversed Rg5-induced cell autophagy and apoptosis in MG-63 cells. Collectively, the current study demonstrated that Rg5 induced human osteosarcoma cell apoptosis through the LC3-mediated autophagy pathway. Under physiological conditions, activation of PI3K/AKT/mTORC1 inhibits LC3 activity and caspase-3-related cell apoptosis. However, Rg5 activated LC3 activity by inhibiting the activation of PI3K/AKT/mTORC1. The present study indicated that Rg5 could be a promising candidate as a chemotherapeutic agent against human osteosarcoma.


2021 ◽  
Vol 14 (6) ◽  
pp. 532
Author(s):  
Muhammad Nazirul Mubin Aziz ◽  
Nurul Fattin Che Rahim ◽  
Yazmin Hussin ◽  
Swee Keong Yeap ◽  
Mas Jaffri Masarudin ◽  
...  

Osteosarcoma (OS) is a life-threatening malignant bone tumor associated with poor prognosis among children. The survival rate of the patient is still arguably low even with intensive treatment provided, plus with the inherent side effects from the chemotherapy, which gives more unfavorable outcomes. Hence, the search for potent anti-osteosarcoma agent with promising safety profile is still on going. Natural occurring substance like curcumin has gained a lot of attention due to its splendid safety profile as well as it pharmacological advantages such as anti-metastasis and anti-angiogenesis. However, natural curcumin was widely known for its poor cellular uptake, which undermines all potential that it possesses. This prompted the development of synthetically synthesized curcuminoid analog, known as (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2- en-1-one (DK1). In this present study, in vitro scratch assay, transwell migration/invasion assay, HUVEC tube formation assay, and ex vivo rat aortic ring assays were performed in order to investigate the anti-metastatic and anti-angiogenic potential of DK1. For further comprehension of DK1 mechanism on human osteosarcoma cell lines, microarray gene expression analysis, quantitative polymerase chain reaction (qPCR), and proteome profiler were adopted, providing valuable forecast from the expression of important genes and proteins related to metastasis and angiogenesis. Based on the data gathered from the bioassays, DK1 was able to inhibit the metastasis and angiogenesis of human osteosarcoma cell lines by significantly reducing the cell motility, number of migrated and invaded cells as well as the tube formation and micro-vessels sprouting. Additionally, DK1 also has significantly regulated several cancer pathways involved in OS proliferation, metastasis, and angiogenesis such as PI3K/Akt and NF-κB in both U-2 OS and MG-63. Regulation of PI3K/Akt caused up-regulation of genes related to metastasis inhibition, namely, PTEN, FOXO, PLK3, and GADD45A. Meanwhile, NF-κB pathway was regulated by mitigating the expression of NF-κB activator such as IKBKB and IKBKE in MG-63, whilst up-regulating the expression of NF-κB inhibitors such as NFKBIA and NFKBIE in U-2 OS. Finally, DK1 also has successfully hindered the metastatic and angiogenic capability of OS cell lines by down-regulating the expression of pro-metastatic genes and proteins like MMP3, COL11A1, FGF1, Endoglin, uPA, and IGFBP2 in U-2 OS. Whilst for MG-63, the significantly down-regulated oncogenes were Serpin E1, AKT2, VEGF, uPA, PD-ECGF, and Endoglin. These results suggest that curcumin analog DK1 may serve as a potential new anti-osteosarcoma agent due to its anti-metastatic and anti-angiogenic attributes.


2021 ◽  
Author(s):  
Hua Wang ◽  
Ji Liang ◽  
Yan Ma ◽  
Lei Zhou ◽  
Yufei Zhang ◽  
...  

Abstract Objectives To establish an accurate, time-saving quality-monitoring method in screening for tumour cell aptamers in order to shorten the screening process and ensure the accurate preparation of the aptamer. Results During quantitative PCR for U2-OS and HOS template, the results showed that the bands obtained from 14 cycles were bright and no non-specific amplification within the optimal template concentrations between 19.0 and 21.0ng/µl. Each round of forward screening was accompanied by reverse screening to accelerate the elimination of non-specific single-strand DNA (ssDNA). In the meanwhile, the aptamer groups were effectively purified specifically bounding to target cells. Besides, we observed that the fluorescence spectroscopy is more accurate, time-saving, and convenient for quality control compared with flow cytometry. Conclusion The method proposed in the study is appropriate for the rapid screening out for human osteosarcoma cell aptamers. The quantitative template concentration, forward screening with back screening, and fluorescence spectroscopy are important methods for accurate preparation and quality control of tumour cell aptamers. It can provide scientific reference data for the amplification of dsDNAs in other sub-libraries.


Life Sciences ◽  
2021 ◽  
Vol 265 ◽  
pp. 118758
Author(s):  
Chia-Chia Chao ◽  
Wei-Fang Lee ◽  
Wei-Hung Yang ◽  
Chih-Yang Lin ◽  
Chien-Kuo Han ◽  
...  

2020 ◽  
Author(s):  
Hua Wang ◽  
Ji Liang ◽  
Yan Ma ◽  
Lei Zhou ◽  
Yufei Zhang ◽  
...  

Abstract Background: We have successfully established an aptamer library for recognition of human osteosarcoma cell using the cell-SELEX method. However, we found that the quality monitoring become a key, resulting in success or failure in the screening process. The study aims to establish an accurate, time-saving quality-monitoring method in screening for tumour cell aptamers in order to shorten the screening process and ensure the accurate preparation of the aptamer. Methods: Two kinds of human osteosarcoma cells (U2-OS, HOS) were selected as the forward screening target cells and human fibrosarcoma cells (HT-1080) as the reverse screening cells to screen the aptamers from the candidate oligonucleotide library. In each round of preparation of the library, PCR was optimised by using quantitative template concentration instead of percentage volume. Each round of forward screening was conducted with reverse screening; Fluorescence spectroscopy and flow cytometry were used to monitor and compare the aptamer libraries. Results: During quantitative PCR for U2-OS and HOS template, the results showed that the bands obtained from 14 cycles were bright and no non-specific amplification within the optimal template concentrations between 19.0 and 21.0ng/µl. Each round of forward screening was accompanied by reverse screening to accelerate the elimination of non-specific single-strand DNA (ssDNA). In the meanwhile, the aptamer groups were effectively purified specifically bounding to target cells. Besides, we observed that the fluorescence spectroscopy is more accurate, time-saving, and convenient for quality control compared with flow cytometry. Conclusion: The method proposed in the study is appropriate for the rapid screening out for human osteosarcoma cell aptamers. The quantitative template concentration, forward screening with back screening, and fluorescence spectroscopy are important methods for accurate preparation and quality control of tumour cell aptamers. It can provide scientific reference data for the amplification of dsDNAs in other sub-libraries.


2020 ◽  
Author(s):  
Hua Wang ◽  
Ji Liang ◽  
Yan Ma ◽  
Lei Zhou ◽  
Yufei Zhang ◽  
...  

Abstract Background: We have successfully developed a novel molecular probe for recognition of human osteosarcoma cell using the cell-SELEX method. The study aims to establish an accurate, time-saving quality-monitoring method in screening for tumour cell adaptors in order to shorten the screening process and ensure the accurate preparation of the adaptor. Methods: Two kinds of human osteosarcoma cells (U2-OS, HOS) were selected as the forward screening target cells and human fibrosarcoma cells (HT-1080) as the reverse screening cells to screen the adaptors from the candidate oligonucleotide library. In each round of preparation of the library, PCR was optimised by using quantitative template concentration instead of percentage volume. Each round of forward screening was conducted with reverse screening; Fluorescence spectroscopy and flow cytometry were used to monitor and compare the aptamer libraries. Results: During quantitative PCR for U2-OS and HOS template, the results showed that the bands obtained from 14 cycles were bright and no non-specific amplification within the optimal template concentrations between 19.0 and 21.0ng/µl. Each round of forward screening was accompanied by reverse screening to accelerate the elimination of non-specific single-strand DNA (ssDNA). In the meanwhile, the adaptor groups were effectively purified specifically bounding to target cells. Besides, we observed that the fluorescence spectroscopy is more accurate, time-saving, and convenient for quality control compared with flow cytometry. Conclusion: The method proposed in the study is appropriate for the rapid screening out for human osteosarcoma cell adaptor. The quantitative template concentration, forward screening with back screening, and fluorescence spectroscopy are important methods for accurate preparation and quality control of tumour cell aptamers. It can provide scientific reference data for the amplification of dsDNAs in other sub-libraries.


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