Simultaneous induction of other types of programmed cell death, alongside apoptosis, in cancer cells may be considered an attractive strategy for the development of more effective anticancer therapies. The present study aimed to investigate the role of AMP‑activated protein kinase (AMPK) in nutrient/serum starvation‑induced necroptosis, which is a programmed form of necrosis, in the presence or absence of p53. The present study detected higher cell proliferation and lower cell death rates in the HCT116 human colon cancer cell line containing a p53 null mutation (HCT116 p53‑/‑) compared with in HCT116 cells harboring wild‑type p53 (HCT116 p53+/+), as determined using a cell viability assay. Notably, western blot analysis revealed a relatively lower level of necroptosis in HCT116 p53‑/‑ cells compared with in HCT116 p53+/+ cells. Investigating the mechanism, it was revealed that necroptosis may be induced in HCT116 p53+/+ cells by significantly increasing reactive oxygen species (ROS) and decreasing mitochondrial membrane potential (MMP), whereas little alterations were detected in HCT116 p53‑/‑ cells. Unexpectedly, a much lower level of ATP was detected in HCT116 p53‑/‑ cells compared with in HCT116 p53+/+ cells. Accordingly, AMPK phosphorylation on the Thr172 residue was markedly increased in HCT116 p53‑/‑ cells. Furthermore, western blot analysis and ROS measurements indicated that AMPK inhibition, using dorsomorphin dihydrochloride, accelerated necroptosis by increasing ROS generation in HCT116 p53‑/‑ cells. However, AMPK activation by AICAR did not suppress necroptosis in HCT116 p53+/+ cells. In conclusion, these data strongly suggested that AMPK activation may be enhanced in HCT116 p53‑/‑ cells under serum‑depleted conditions via a drop in cellular ATP levels. In addition, activated AMPK may be at least partially responsible for the inhibition of necroptosis in HCT116 p53‑/‑ cells, but not in HCT116 p53+/+cells.
The capability to detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and identify immune responses among the population is crucial for managing the outbreak of the COVID-19 pandemic. Although PCR-based nucleic acid detection techniques are utilized to detect viral infection in people, alternative tests capable of distinguishing between exposure and infection are urgently needed beyond this restricted window of detectable viral replication. Antibodies are produced in human sera within a few days after viral infection, providing longer period for performing tests to acquire reliable database. Herewith, we provide the results of our in-house developed ELISA (Enzyme-Linked Immunosorbent Assay) that displays all of the properties necessary for high-throughput of human sera sample analysis. This test does not involve the handling of live viruses, although it detects a variety of antibody types in serum and plasma of human after exposure to the virus. For in-house development of the kit, the nucleocapsid (N) gene of SARS-CoV-2 virus was cloned in the prokaryotic expression vector pGEX-6P-1, and purified N protein was used to detect IgG antibodies in human sera samples. In total 76 human serum samples that were collected before novel coronavirus registry in Mongolia in March 2020, as well as 200 serum samples from patients who had been infected by SARS-CoV-2 virus, were used. Among 200 serum samples, 188 were positive and 12 were false negative, while in non-infected cases 69 were negative and 7 were false positive, suggesting 94 per cent sensitivity and 90.7 per cent specificity of the kit, with p-values of 0.02.
Currently, many available anti-cancer therapies are targeting apoptosis. However, many cancer cells have acquired resistance to apoptosis. To overcome this problem, simultaneous induction of other types of programmed cell death in addition to apoptosis of cancer cells might be an attractive strategy. For this purpose, we initially investigated the inhibitory role of TRIP-Br1/XIAP in necroptosis, a regulated form of necrosis, under nutrient/serum starvation. Our data showed that necroptosis was significantly induced in all tested 9 different types of cancer cell lines in response to prolonged serum starvation. Among them, necroptosis was induced at a relatively lower level in MCF-7 breast cancer line that was highly resistant to apoptosis than that in other cancer cell lines. Interestingly, TRIP-Br1 oncogenic protein level was found to be very high in this cell line. Upregulated TRIP-Br1 suppressed necroptosis by repressing reactive oxygen species generation. Such suppression of necroptosis was greatly enhanced by XIAP, a potent inhibitor of apoptosis. Our data also showed that TRIP-Br1 increased XIAP phosphorylation at serine87, an active form of XIAP. Our mitochondrial fractionation data revealed that TRIPBr1 protein level was greatly increased in the mitochondria upon serum starvation. It suppressed the export of CypD, a vital regulator in mitochondria-mediated necroptosis, from mitochondria to cytosol. TRIP-Br1 also suppressed shikoninmediated necroptosis, but not TNF-α-mediated necroptosis, implying possible presence of another signaling pathway in necroptosis. Taken together, our results suggest that TRIPBr1/XIAP can function as onco-proteins by suppressing necroptosis of cancer cells under nutrient/serum starvation.
// Samil Jung 1, * , Hyung-In Moon 2, * , Beom Suk Lee 1, * , Subeen Kim 1 , Nguyen Thi Ngoc Quynh 1 , Jimin Yu 2 , Dan-Diem Thi Le 1 , Zolzaya Sandag 1 , Hyegyeong Lee 1 , Hyojeong Lee 1 , Nguyen Hai Anh 1 , Young Yang 1 , Jong-Seok Lim 1 , Keun-Il Kim 1 and Myeong-Sok Lee 1 1 Department of Biological Science, Sookmyung Women's University, Seoul, 14310, South Korea 2 Department of Medicinal Biotechnology, College of Health Sciences, Dong-A University, Busan, 49315, South Korea * These authors have contributed equally to this work Correspondence to: Myeong-Sok Lee, email: mslee@sookmyung.ac.kr Keywords: Angelica amurensis; cis-khellactone; anti-cancer drug; apoptosis, autophagy-mediated cell death; necrosis/necroptosis Received: September 19, 2017 Accepted: February 27, 2018 Published: March 30, 2018 ABSTRACT Angelica amurensis has traditionally been used to treat various medical problems. In this report, we introduce cis-khellactone as a new anti-cancer agent, which was isolated from the chloroform soluble fraction of the rhizomes of Angelica amurensis . Its anti-cancerous effect was at first tested in MCF7 and MDA-MB-231 breast cell lines, in which MCF7 is well known to be resistant to many anti-cancer drugs; MCF10A normal breast cell line was used as a control. In vitro experiments showed that cis-khellactone suppressed cell growth and proliferation at a relatively low concentrations (<5 μg/ml) and decreased cell viability at high concentrations (>10 μg/ml) in both cancer cell lines in a time- and concentration-dependent manner. This anti-cancerous effect was also checked in additional 16 different types of normal and cancer cell lines. Cis-khellactone treatment significantly suppressed cell proliferation and enhanced cell death in all tested cancer cell lines. Furthermore, Western blot analysis showed that cis-khellactone induced three types of programmed cell death (PCD): apoptosis, autophagy-mediated cell death, and necrosis/necroptosis. Cis-khellactone concentration-dependently decreased cell viability by increasing the level of reactive oxygen species (ROS) and decreasing mitochondrial membrane potential (MMP), which are related to all three types of PCD. Mitochondrial fractionation data revealed that cis-khellactone induced the translocation of BAX and BAK into mitochondria as well as the overexpression of VDAC1, which probably accelerates MMP disruption and finally cell death. Importantly, our extended in vivo studies with xenograft model further confirmed these findings of anti-cancerous effects and showed no harmful effects in normal tissues, suggesting that there would be no side effects in humans.
Hippophae rhamnoides L., which belongs to the Elaegnaceae family, is one of the medically and environmentally valuable berry crops with its high nutritious and bioactive compounds. Despite its high demand in the food, medicinal and agricultural industries, this species has been less studied molecularly. In view of this, an effort has been made in the present study to characterize 24 accessions of H. rhamnoides collected from different geographical regions of Mongoliaa through random amplified polymorphic DNA (RAPD) markers. A total of 10 RAPD primers were used in the present study for their ability to produce clear, scorable amplicons. The RAPD analysis totally generated 87 bands, of which 84 (96.34%) were polymorphic, pointing to a high degree of genetic variation. The similarity coefficient ranged from 0.4-1 with the mean of 0.78. The UPGMA dendrogram was generated using these data grouped accessions into two main clusters. Cluster analysis reflected a relatively close relationship between accessions grown at the same or neighbouring areas. Thus, our data could be informative for further selection and management of germplasm collections and crossing strategies for sea buckthorn.
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