The effects of EGCG on the selective death of cancer cells by modulating antioxidant pathways through autophagy were explored in various normal and cancer cells. EGCG positively regulated the p62-KEAP1-NRF2-HO-1 pathway in normal cells, while negatively regulating it in cancer cells, leading to selective apoptotic death of cancer cells. In EGCG-treated MRC5 cells (EGCG-MRC5), autophagic flux was blocked, which was accompanied by the formation of p62-positive aggregates. However, EGCG-treated HeLa cells (EGCG-HeLa) showed incomplete autophagic flux and no aggregate formation. The levels of P-ULK1 S556 and S758 increased in EGCG-MRC5 through AMPK-mTOR cooperative interaction. In contrast, EGCG treatment in HeLa cells led to AMPK-induced mTOR inactivation, resulting in abrogation of P-ULK1 S556 and S758 levels. AMPK knockout in EGCG-HeLa restored positive regulation of the p62-mediated pathway, which was accompanied by increased P-mTOR S2448 and P-ULK1 S758 levels. Knockdown of 67LR in EGCG-HeLa abolished AMPK activity but did not restore the p62-mediated pathway. Surprisingly, both AMPK knockout and 67LR knockdown in EGCG-HeLa markedly increased cell viability, despite differential regulation of the antioxidant enzyme HO-1. In conclusion, EGCG induces the selective death of cancer cells through the modulation of at least two autophagy-dependent and independent regulatory pathways: negative regulation involves the mTOR-ULK1 (S556 and S758)-p62-KEAP1-NRF2-HO-1 axis via AMPK activation, whereas positive regulation occurs through the 67LR-AMPK axis.

Microtubule acetylation has been shown to regulate actin filament dynamics by modulating signaling pathways that control actin organization, although the precise mechanisms remain unknown. In this study, we found that the downregulation of microtubule acetylation via the disruption ATAT1 (which encodes α-tubulin N-acetyltransferase 1) inhibited the expression of RhoA, a small GTPase involved in regulating the organization of actin filaments and the formation of stress fibers. Analysis of RHOA promoter and chromatin immunoprecipitation assays revealed that C/EBPβ is a major regulator of RHOA expression. Interestingly, the majority of C/EBPβ in ATAT1 knockout (KO) cells was found in the nucleus as a 27-kDa fragment (referred to as C/EBPβp27) lacking the N-terminus of C/EBPβ. Overexpression of a gene encoding a C/EBPβp27-mimicking protein via an N-terminal deletion in C/EBPβ led to competitive binding with wild-type C/EBPβ at the C/EBPβ binding site in the RHOA promoter, resulting in a significant decrease of RHOA expression. We also found that cathepsin L (CTSL), which is overexpressed in ATAT1 KO cells, is responsible for C/EBPβp27 formation in the nucleus. Treatment with a CTSL inhibitor led to the restoration of RHOA expression by downregulation of C/EBPβp27 and the invasive ability of ATAT1 KO MDA-MB-231 breast cancer cells. Collectively, our findings suggest that the downregulation of microtubule acetylation associated with ATAT1 deficiency suppresses RHOA expression by forming C/EBPβp27 in the nucleus through CTSL. We propose that CTSL and C/EBPβp27 may represent a novel therapeutic target for breast cancer treatment. [BMB Reports 2024; 57(6): 293-298].

Over the past few decades, microtubules have been targeted by various anticancer drugs, including paclitaxel and eribulin. Despite their promising effects, the development of drug resistance remains a challenge. We aimed to define a novel cell death mechanism that targets microtubules using eribulin and to assess its potential in overcoming eribulin resistance. Notably, treating non-resistant breast cancer cells with eribulin led to increased microtubule acetylation around the nucleus and cell death. Conversely, eribulin-resistant (EriR) cells did not exhibit a similar increase in acetylation, even at half-maximal inhibitory concentrations. Interestingly, silencing the ATAT1 gene, which encodes the α-tubulin N-acetyltransferase 1 (the enzyme responsible for microtubule acetylation), induces eribulin resistance, mirroring the phenotype of EriR cells. Moreover, eribulin-induced acetylation of microtubules facilitates the transport of Ca2+ from the ER to the mitochondria, releasing cytochrome c and subsequent cell death. Transcriptome analysis of EriR cells revealed a significant downregulation of ER stress-induced apoptotic signals, particularly the activity of protein kinase RNA-like ER kinase (PERK), within the unfolded protein response signaling system. Pharmacological induction of microtubule acetylation through a histone deacetylase 6 inhibitor combined with the activation of PERK signaling using the PERK activator CCT020312 in EriR cells enhanced mitochondrial Ca2+ accumulation and subsequent cell death. These findings reveal a novel mechanism by which eribulin-induced microtubule acetylation and increased PERK activity lead to Ca2+ overload from the ER to the mitochondria, ultimately triggering cell death. This study offers new insights into strategies for overcoming resistance to microtubule-targeting agents.

Matrix stiffness has been shown to play a critical role in cancer progression by influencing various cellular processes, including epidermal growth factor (EGF) signaling. However, the underlying molecular mechanisms are not fully understood. Here, we investigated the role of adaptor-related protein complex 1 subunit sigma 1 (AP1S1), a component of adaptor protein complex-1, in the regulation of EGF receptor (EGFR) intracellular trafficking during cancer cell progression. We found that AP1S1 expression was upregulated under stiff matrix conditions, resulting in the regulation of EGFR trafficking in non-small cell lung adenocarcinoma cells. Knockout of AP1S1 caused the lysosomal degradation of EGFR, leading to suppressed EGF-induced anaplastic lymphoma receptor tyrosine kinase phosphorylation. In addition, the downregulation of AP1S1 increased the sensitivity of H1975 cancer cells, which are resistant to tyrosine kinase inhibitors, to erlotinib. Collectively, our results suggest that AP1S1 could regulate EGFR recycling under stiff matrix conditions, and AP1S1 inhibition could be a novel strategy for treating cancer cells resistant to EGFR-targeted anticancer drugs.

Excessive production and accumulation of amyloid‐beta (Aβ) in the brain are one of the hallmarks of Alzheimer's disease (AD). Although oxidative stress is known to trigger and promote the progression of AD, the molecular relationship between oxidative stress and Aβ production is not yet fully understood. In this study, we demonstrate that microtubule acetylation induced by oxidative stress plays a critical role in Aβ production and secretion by altering the subcellular distribution of Aβ precursor protein (APP)‐containing lysosomal vesicles. Under oxidative stress, both H4‐APP Swe/Ind and HEK293T‐APP Swe/Ind cell lines showed increased microtubule acetylation and Aβ secretion. Knockdown (KD) of alpha‐tubulin N‐acetyltransferase 1 ( ATAT1 ) by using a lentiviral shRNA not only inhibited the generation of intermediate APP fragments, such as β‐CTF and AICD, but also suppressed Aβ secretion. Oxidative stress promoted the dispersion of LAMP1‐positive vesicles to the periphery of the cell through microtubule acetylation, leading to the formation of neutralized lysosomal vesicles (NLVs), which was inhibited by ATAT1 KD. Treatment of the cells with the dynein ATPase inhibitor EHNA or downregulation of LIS1, a regulator of dynein‐mediated intracellular transport, increased the peripheral localization of NLVs and promoted Aβ secretion, whereas KD of ADP ribosylation factor like GTPase 8B showed the opposite result. ATAT1 KD in the hippocampal region of the 5×FAD AD mouse model also showed significant reductions in Aβ plaque accumulation and memory loss. Taken together, these findings suggest that oxidative stress–induced microtubule acetylation promotes the peripheral localization of lysosomal vesicles to form NLVs, thereby enhancing Aβ secretion.

Karyopherin-α3 (KPNA3), a karyopherin- α isoform, is intimately associated with metastatic progression via epithelial-mesenchymal transition (EMT). However, the molecular mechanism underlying how KPNA3 acts as an EMT inducer remains to be elucidated. In this report, we identified that KPNA3 was significantly upregulated in cancer cells, particularly in triple-negative breast cancer, and its knockdown resulted in the suppression of cell proliferation and metastasis. The comprehensive transcriptome analysis from KPNA3 knockdown cells indicated that KPNA3 is involved in the regulation of numerous EMTrelated genes, including the downregulation of GATA3 and E-cadherin and the up-regulation of HAS2. Moreover, it was found that KPNA3 EMT-mediated metastasis can be achieved by TGF-β or AKT signaling pathways; this suggests that the novel independent signaling pathways KPNA3-TGF-β-GATA3-HAS2/E-cadherin and KPNA3-AKT-HAS2/E-cadherin are involved in the EMT-mediated progress of TNBC MDA-MB-231 cells. These findings provide new insights into the divergent EMT inducibility of KPNA3 according to cell and cancer type. [BMB Reports 2023; 56(2): 120-125].

During aggressive cancer progression, cancer cells adapt to unique microenvironments by withstanding various cellular stresses, including endoplasmic reticulum (ER) stress. However, the mechanism whereby cancer cells overcome the ER stress to survive remains to be elucidated. Herein, we demonstrated that microtubule acetylation in cancer cells grown on a stiff matrix promotes cancer progression by preventing excessive ER stress. Downregulation of microtubule acetylation using shRNA or CRSIPR/Cas9 techniques targeting ATAT1, which encodes α-tubulin N-acetyltransferase (αTAT1), resulted in the upregulation of ER stress markers, changes in ER morphology, and enhanced tunicamycin-induced UPR signaling in cancer cells. A set of genes involved in cancer progression, especially focal adhesion genes, were downregulated in both ATAT1-knockout and tunicamycin-treated cells, whereas ATAT1 overexpression restored the gene expression inhibited by tunicamycin. Finally, the expression of ATAT1 and ER stress marker genes were negatively correlated in various breast cancer types. Taken together, our results suggest that disruption of microtubule acetylation is a potent therapeutic tool for preventing breast cancer progression through the upregulation of ER stress. Moreover, ATAT1 and ER stress marker genes may be useful diagnostic markers in various breast cancer types.

Established genetic risk factors for Alzheimer’s disease (AD) account for only a portion of AD heritability. The aim of this study was to identify novel associations between genetic variants and AD-specific brain atrophy. We conducted genome-wide association studies for brain magnetic resonance imaging measures of hippocampal volume and entorhinal cortical thickness in 2643 Koreans meeting the clinical criteria for AD ( n = 209), mild cognitive impairment ( n = 1449) or normal cognition ( n = 985). A missense variant, rs77359862 (R274W), in the SHANK-associated RH Domain Interactor ( SHARPIN ) gene was associated with entorhinal cortical thickness ( p = 5.0 × 10 −9 ) and hippocampal volume ( p = 5.1 × 10 −12 ). It revealed an increased risk of developing AD in the mediation analyses. This variant was also associated with amyloid-β accumulation ( p = 0.03) and measures of memory ( p = 1.0 × 10 −4 ) and executive function ( p = 0.04). We also found significant association of other SHARPIN variants with hippocampal volume in the Alzheimer’s Disease Neuroimaging Initiative (rs3417062, p = 4.1 × 10 −6 ) and AddNeuroMed (rs138412600, p = 5.9 × 10 −5 ) cohorts. Further, molecular dynamics simulations and co-immunoprecipitation indicated that the variant significantly reduced the binding of linear ubiquitination assembly complex proteins, SHPARIN and HOIL-1 Interacting Protein (HOIP), altering the downstream NF-κB signaling pathway. These findings suggest that SHARPIN plays an important role in the pathogenesis of AD.

Spatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (βPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying βPix-mediated cancer cell invasion remain unclear. The clinical significance of βPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for βPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of βPix on CRC progression. A βPix-SH3 antibody delivery system was used to determine the effects of the βPix-Dyn2 complex in CRC cells. We found that the Src homology 3 (SH3) domain of βPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The βPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of βPix enhanced βPix-Dyn2 complex formation. Disruption of the βPix-Dyn2 complex by βPix-SH3 antibodies targeting intracellular βPix inhibited CRC cell invasion. Our data indicate that spatiotemporal regulation of the Src-βPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the βPix-Dyn2 complex may be a useful therapeutic strategy for CRC.

Background Procollagen type I N-terminal propeptide (PINP) is one of the most clinically useful bone formation biomarkers. Therefore, the purpose of this study was to independently evaluate the performance of automated total PINP assay and established age- and gender-specific reference intervals for PINP in healthy Korean population. Methods The imprecision, linearity, and detection capability of Elecsys total PINP assay was determined and reference interval was established using 599 serums from Korean population with normal bone mineral densities based on bone densitometry. Age groups were divided into 20s, 30s, 40s, 50s, 60s and over. Results Elecsys total PINP had excellent performance in imprecision, linearity, and detection capability. When partitioning age groups in Korean male and female populations, there was significant difference in total PINP between different age groups. In male populations, PINP level was decreased with increasing age, then it remained steady after middle-age. In female populations, there was a decreasing tendency similar to that in the male population with a sharp increase in the 50 to 59 age group. Conclusions Elecsys total PINP assay showed precise and reliable performance in our study. We established age-related PINP reference intervals for Korean male and female population with normal bone mineral densities.

Members of the Mongol imperial family (designated the Golden family) are buried in a secret necropolis; therefore, none of their burial grounds have been found. In 2004, we first discovered 5 graves belonging to the Golden family in Tavan Tolgoi, Eastern Mongolia. To define the genealogy of the 5 bodies and the kinship among them, SNP and/or STR profiles of mitochondria, autosomes, and Y chromosomes were analyzed. Four of the 5 bodies were determined to carry the mitochondrial DNA haplogroup D4, while the fifth carried haplogroup CZ, indicating that this individual had no kinship with the others. Meanwhile, Y-SNP and Y-STR profiles indicate that the males examined belonged to the R1b-M343 haplogroup. Thus, their East Asian D4 or CZ matrilineal and West Eurasian R1b-M343 patrilineal origins reveal genealogical admixture between Caucasoid and Mongoloid ethnic groups, despite a Mongoloid physical appearance. In addition, Y chromosomal and autosomal STR profiles revealed that the four D4-carrying bodies bore the relationship of either mother and three sons or four full siblings with almost the same probability. Moreover, the geographical distribution of R1b-M343-carrying modern-day individuals demonstrates that descendants of Tavan Tolgoi bodies today live mainly in Western Eurasia, with a high frequency in the territories of the past Mongol khanates. Here, we propose that Genghis Khan and his family carried Y-haplogroup R1b-M343, which is prevalent in West Eurasia, rather than the Y-haplogroup C3c-M48, which is prevalent in Asia and which is widely accepted to be present in the family members of Genghis Khan. Additionally, Tavan Tolgoi bodies may have been the product of marriages between the lineage of Genghis Khan’s Borjigin clan and the lineage of either the Ongud or Hongirad clans, indicating that these individuals were members of Genghis Khan’s immediate family or his close relatives.

Background: To prevent the recurrence of genitourinary infections and to reduce the risks of their complications, accurate and rapid diagnosis are required. STDetect (R) Chip is a DNA chip which allows for the simultaneous detection of 13 major genitourinary pathogens in a single vaginal swab or urine specimen. We evaluated the analytical performance of the STDetect (R) Chip for detecting target pathogens that commonly cause genitourinary infections.Methods: The target pathogens of the STDetect (R) Chip are Chlamydia trachomatis, Candida albicans, Enterococcus faecalis, Gardnerella vaginalis, Mycoplasma hominis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Staphylococcus aureus, Klebsiella pneumoniae, Trichomonas vaginalis, and Herpes simplex virus types 1 and 2. Performance of the STDetect (R) Chip for the detection of target pathogens was evaluated comparing with the result of direct sequencing and conventional multiplex PCR assay. And precision tests for STDetect (R) Chip were performed with quality control materials.Results: The STDetect (R) Chip showed high sensitivities (95.1%-100%), specificities (93.4% to 100%), concordance rates (95.0%-100%), positive predictive values (69.8%-100%), and negative predictive values (93.1%-100%) in its identification of 13 target pathogens. The STDetect (R) Chip had a particularly excellent concordance rate (965%) for the 4 major pathogens, C albicans, G. vaginalis, M. hominis, and U. urealyticum, compared with direct sequencing. Comparing to multiplex PCR assay, STDetect (R) Chip showed better sensitivity for detecting M. hominis (97.0% vs. 54.5%) and U. urealyticum (932% vs. 65.9%). In precision tests, coefficients of variations for signal intensities were ranged from 11.2% to 26.2%.Conclusion: The STDetect (R) Chip showed excellent analytical performance, and its result was in good agreement with that obtained by direct sequencing. (C) 2014 Elsevier B.V. All rights reserved.

(-)-Epigallocatechin-3-gallate (EGCG) induces apoptosis in cancer cells without adversely affecting normal cells. Understanding the cancer-specific cytotoxic activity of EGCG is very important in defining the mechanism of tumorigenesis and identifying superb chemotherapeutic agents against cancer. We comparatively assayed human telomerase reverse transcriptase (hTERT)-mediated apoptosis by EGCG-induced reactive oxygen species (ROS) in normal cells and cancer cells. EGCG showed differential levels of ROS induction between the cell types; ROS, especially hydrogen peroxide, was highly induced in cancer cells, while it was not in normal cells. In addition, the higher level of ROS down-regulated hTERT via binding of CCCTC binding factor (CTCF) to the core promoter region of hTERT, which repressed hTERT expression. CTCF binding was epigenetically controlled by the demethylation of the previously hypermethylated site for CTCF, which was induced by down-regulation of DNA methyltransferase 1 (DNMT1). In contrast, hTERT down-regulation was not observed in normal cells. These results suggest that preferential death of cancer cells by EGCG could be caused by the cancer-specific induction of ROS and epigenetic modulation of expression of apoptosis-related genes, such as hTERT.

BACKGROUND:Human immunodeficiency virus type-1 (HIV-1) RNA viral load is a surrogate marker that is routinely used to determine indications for, and monitor the effectiveness of HIV-1 treatment. We developed three reagents for potential use in routine quality control of HIV-1 RNA quantitative assays. In this report, we compare the stability of these re-agents in storage and compare their performance in three different HIV-1 RNA quantitative assays.METHODS:The candidate reagents were derived from readily available pre-existing reagents and examined for stability at different storage temperatures. They were compared in three commercially available HIV-1 RNA quantitative assays: the Cobas TaqMan HIV-1 Test (Cobas TaqMan), the RealTime HIV-1 Assay (Abbott RealTime), and the NucliSens EasyQ HIV-1 Assay v1.1 (NucliSens EasyQ).RESULTS:The candidate reagent derived from an HIV culture supernatant (candidate CS) was the most stable of the three candidates and showed good reproducibility. Candidate CS yielded the highest HIV-1 titer of the three candidates in the Cobas TaqMan assay and the lowest HIV-1 titer and stability of the three candidates in the NucliSens EasyQ system.CONCLUSIONS:The candidate CS is the most appropriate of the three candidate reagents for quantitative testing of HIV-1 RNA. This working reagent should be useful for use in routine calibration for quality control in centers with limited financial resources. The Cobas TaqMan assay tended to yield higher viral load results than the other assays when used with our three candidate reagents.

We have performed analyses using ancient DNA extracted from 25 excavated human bones, estimating around the 1(st) century B.C. Ancient human bones were obtained from Nukdo Island, which is located off of the Korean peninsula of East Asia. We made concerted efforts to extract ancient DNA of high quality and to obtain reproducible PCR products, as this was a primary consideration for this extensive kind of undertaking. We performed PCR amplifications for several regions of the mitochondrial DNA, and could determine mitochondrial haplogroups for 21 ancient DNA samples. Genetic information from mitochondrial DNA belonged to super-haplogroup M, haplogroup D or its sub-haplogroups (D4 or D4b), which are distinctively found in East Asians, including Koreans or Japanese. The dendrogram and principal component analysis based on haplogroup frequencies revealed that the Nukdo population was close to those of the East Asians and clearly distinguished from populations shown in the other regions. Considering that Nukdo is geologically isolated in the southern part of them Korean peninsula and is a site of commercial importance with neighboring countries, these results may reflect genetic continuity for the habitation and migration of ethnic groups who had lived in a particular area in the past. Therefore, we suggest that phylogenetic analyses of ancient DNA have significant advantages for clarifying the origins and migrations of ethnic groups, or human races.

Interferon (INF) is an effective drug in treating several human diseases. Ge-132, which is the most common and well-studied organic germanium, has been reported to induce INF-gamma and has undergone clinical trials with promising preclinical results. However, it has been reported that long-term ingestion or high doses of organic Ge-132 causes similar toxic effects as GeO(2) because Ge-132 can be easily contaminated with significant amounts of inorganic germanium during the preparation. In this study, we synthesized the water-soluble organogermanium compound (Ge-OH) without possible contamination with toxic inorganic germanium and showed that Ge-OH is a better INF-gamma inducer than Ge-132 by an animal study.

Background: Although many single nucleotide polymorphisms (SNPs) of mtDNA h ave been found to be associated with type 2 diabetes mellitus, the results of studies using different population samples and different methods are mixed. Therefore, we conducted a genetic association study of mtDNA SNPs and type 2 diabetes mellitus in a Korean sample and compared our results with those of studies co nducted in other human populations. Methods: A total of 298 blood samples from 147 type 2 diabetic patients and 151 normal controls were surveyed for SNPs via PCR directed sequencing. Sequencing analy ses were performed using the SeqMan module of the DNASTAR program. The identified SNPs were compared to previously reported SNP lists on NCBI and V-mitoSNP. Results: A total of 24 SNPs were identified in the MT-RNR2, MR-TL1 and MT-ND1 mtDNA genes in Korean type 2 diabetes mellitus patients and normal controls. The SNPs identified in the Korean sample were not closely associated with the type 2 diabetes mellitus phenotype, a significantly different result from those previously observed in European, Chinese and Japanese samples. Additionally, a haplotype and prevalence analysis could not detect any differences between the type 2 diabetes mellitus patients and normal controls. Conclusion: The 24 mtDNA SNPs were not associ ated with type 2 diabetes mellitus risk in our Korean sample. The results of the present study support the possibilit y that mtDNA SNPs have a differential effect on the risk of type 2 diabetes mellitus according to geographical origin. (Korean Diabetes J 33:382-391, 2009)

Since nonsense mutations are closely associated with severe conditions of genetic disorders, including familial cancers, rapid and precise detection of those mutations is very important for research purposes and molecular diagnosis. Currently, screening methods such as the FASAY (functional analysis of separated alleles in yeast) and the Y‐SC (stop codon assay in yeast) are used for functional detection of nonsense mutations in genes of interest. But these yeast‐based approaches are time‐consuming, expensive and complicated. In order to circumvent these problems, we, in the present study, devised a novel Escherichia coli‐based screening method, the E‐SC (E. coli stop codon assay) for the detection of heterozygous nonsense mutations in genes of interest. Our strategy was based on the fact that the plasmid replicating with a low copy number in E. coli allows an effective separation of normal and mutant alleles. Moreover, it relies on the expression vector, resulting in the formation of white and blue colonies for mutant and normal alleles through the expression of PCR‐amplified fragment/lacZ fusion protein respectively. To evaluate the applicability of the E‐SC method for the detection of the heterozygous truncating mutation, PCR‐amplified exon 7 of the StAR [steroidogenic acute regulatory protein; causative gene of the CAH (congenital lipoid adrenal hyperplasia)] and RT (reverse transcription)–PCR‐amplified full‐length cDNA of MeCP2 (methyl‐CpG‐binding protein 2; causative gene of Rett syndrome) were used. The E‐SC showed an almost 1:1 ratio of blue/white colonies in all patients examined, whereas the control samples produced blue colonies only. These results demonstrate that the E‐SC system is useful for rapid and precise detection of known and unknown heterozygous truncation mutations in genes that cause genetic disorders and familial cancers.