EGCG-induced selective death of cancer cells through autophagy-dependent regulation of the p62-mediated antioxidant survival pathway.

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.

Enhancing Lung Cancer Classification through Integration of Liquid Biopsy Multi-Omics Data with Machine Learning Techniques.

Early detection of lung cancer is crucial for patient survival and treatment. Recent advancements in next-generation sequencing (NGS) analysis enable cell-free DNA (cfDNA) liquid biopsy to detect changes, like chromosomal rearrangements, somatic mutations, and copy number variations (CNVs), in cancer. Machine learning (ML) analysis using cancer markers is a highly promising tool for identifying patterns and anomalies in cancers, making the development of ML-based analysis methods essential. We collected blood samples from 92 lung cancer patients and 80 healthy individuals to analyze the distinction between them. The detection of lung cancer markers Cyfra21 and carcinoembryonic antigen (CEA) in blood revealed significant differences between patients and controls. We performed machine learning analysis to obtain AUC values via Adaptive Boosting (AdaBoost), Multi-Layer Perceptron (MLP), and Logistic Regression (LR) using cancer markers, cfDNA concentrations, and CNV screening. Furthermore, combining the analysis of all multi-omics data for ML showed higher AUC values compared with analyzing each element separately, suggesting the potential for a highly accurate diagnosis of cancer. Overall, our results from ML analysis using multi-omics data obtained from blood demonstrate a remarkable ability of the model to distinguish between lung cancer and healthy individuals, highlighting the potential for a diagnostic model against lung cancer.

Advances in methylation analysis of liquid biopsy in early cancer detection of colorectal and lung cancer.

Methylation patterns in cell-free DNA (cfDNA) have emerged as a promising genomic feature for detecting the presence of cancer and determining its origin. The purpose of this study was to evaluate the diagnostic performance of methylation-sensitive restriction enzyme digestion followed by sequencing (MRE-Seq) using cfDNA, and to investigate the cancer signal origin (CSO) of the cancer using a deep neural network (DNN) analyses for liquid biopsy of colorectal and lung cancer. We developed a selective MRE-Seq method with DNN learning-based prediction model using demethylated-sequence-depth patterns from 63,266 CpG sites using SacII enzyme digestion. A total of 191 patients with stage I-IV cancers (95 lung cancers and 96 colorectal cancers) and 126 noncancer participants were enrolled in this study. Our study showed an area under the receiver operating characteristic curve (AUC) of 0.978 with a sensitivity of 78.1% for colorectal cancer, and an AUC of 0.956 with a sensitivity of 66.3% for lung cancer, both at a specificity of 99.2%. For colorectal cancer, sensitivities for stages I-IV ranged from 76.2 to 83.3% while for lung cancer, sensitivities for stages I-IV ranged from 44.4 to 78.9%, both again at a specificity of 99.2%. The CSO model's true-positive rates were 94.4% and 89.9% for colorectal and lung cancers, respectively. The MRE-Seq was found to be a useful method for detecting global hypomethylation patterns in liquid biopsy samples and accurately diagnosing colorectal and lung cancers, as well as determining CSO of the cancer using DNN analysis.Trial registration: This trial was registered at ClinicalTrials.gov (registration number: NCT04253509) for lung cancer on 5 February 2020, https://clinicaltrials.gov/ct2/show/NCT04253509 . Colorectal cancer samples were retrospectively registered at CRIS (Clinical Research Information Service, registration number: KCT0008037) on 23 December 2022, https://cris.nih.go.kr , https://who.init/ictrp . Healthy control samples were retrospectively registered.

Selective death of cancer cells by preferential induction of reactive oxygen species in response to (-)-epigallocatechin-3-gallate.

(-)-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.

TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116.

Trichostatin A (TSA), an inhibitor of histone deacetylase, is a well-known antitumor agent that effectively and selectively induces tumor growth arrest and apoptosis. Recently, it was reported that hTERT is one of the primary targets for TSA-induced apoptosis in cancer cells but the mechanism of which has not yet been elucidated. In the present study, to better understand the epigenetic regulation mechanism responsible for the repression of hTERT by TSA, we examined expression of hTERT in the HCT116 colon cancer cell line after treatment with TSA and performed site-specific CpG methylation analysis of the hTERT promoter. We found that TSA-induced the demethylation of site-specific CpGs on the promoter of hTERT, which was caused by down-regulation of DNA methyltransferase 1 (DNMT1). Among the demethylated region, the 31st-33rd CpGs contained a binding site for CTCF, an inhibitor of hTERT transcription. ChIP analysis revealed that TSA-induced demethylation of the 31st-33rd CpGs promoted CTCF binding on hTERT promoter, leading to repression of hTERT. Taken together, down-regulation of DNMT1 by TSA caused demethylation of a CTCF binding site on the hTERT promoter, the result of which was repression of hTERT via recruitment of CTCF to the promoter.

Akt1 inhibition by RNA interference sensitizes human non-small cell lung cancer cells to cisplatin.

Akt/protein kinase B signaling is very important for cancer cell survival and growth when cells are exposed to various apoptotic stimuli. Akt is constitutively activated in NSCLC cells and is a potential target for enhancing the cytotoxicity of chemotherapeutic agents in treatment of NSCLC. In our study, we investigated whether down-regulating Akt1 using RNAi techniques can enhance sensitivity to cisplatin in NSCLC cells. An siRNA targeting Akt1 significantly decreased the protein level of Akt1 and the activity of ERK. Treatment of these cells with 20 muM cisplatin increased apoptotic cell death approximately 2.6-fold compared to cells transfected with a scrambled siRNA. While Akt activity was slightly reduced, ERK activity was greatly increased in cells treated with cisplatin alone. Pretreatment of these cells with the selective MEK inhibitor U0126 effectively reduced the level of cisplatin-induced apoptosis. These results imply that cisplatin-induced MEK/ERK activation appears to mediate apoptotic cell death, but that constitutively activated Akt1 and/or ERK pathway may mediate resistance to cisplatin in NSCLC cells. Taken together, our data demonstrate that down-regulation of Akt1 using RNAi enhances the chemosensitivity of NSCLC cells to cisplatin.

Inhibition of Importin ß1 With a 2-Aminothiazole Derivative Resulted in G2/M Cell-cycle Arrest and Apoptosis.

BACKGROUND: The design and synthesis of novel chemotherapeutic agents that can induce apoptosis and cell-cycle arrest has emerged as an attractive approach for the treatment of cancer, because they can limit possible nonspecific effects of compound treatment. Previous studies established that the expression of KPNB1 was increased in several cancer cells and transformed cell lines and inhibition of KPNB1 using siRNA significantly inhibited cervical tumour proliferation, but did not affect normal cervical epithelium. Recently, we reported that a KPNB1 inhibitor, the 2-aminothiazole derivative 1, possesses strong anti-proliferative effects against several cancer cells in the nanomolar concentration range. RESULTS: Treatment with compound 1 interferes with cell-cycle progression in the G2/M phase, as detected by flow cytometry analysis and results in apoptosis by the intrinsic pathway. Fluorescence microscopic analysis of mitotic cells predominantly mitotic abnormal cells with monopolar spindles and treatment with compound 1 did not affect polymerization of microtubules. CONCLUSION: Compound 1, as a KPNB1 inhibitor, might be a good target for future development of anticancer agents showing the activities of apoptosis and cell cycle arrest.

Molecular Genealogy of a Mongol Queen's Family and Her Possible Kinship with Genghis Khan.

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.

Molecular genetic characterization of a Korean split hand/split foot malformation (SHFM).

Split hand/split foot malformation (SHFM; ectrodactyly) is genetically heterogeneous, with mutations identified at five loci (SHFM1 at 7q21.3, SHFM2 at Xq26, SHFM3 at 10q24, SHFM4 at 3q27 and SHFM5 at 2q31). In this study, we attempted to identify and localize the causative allele of a Korean case of SHFM. Pedigree analysis showed that the Korean SHFM was autosomally dominant and its penetrance was high, indicating that it was not caused by SHFM2. Clinical features were variable, but limited to the four limbs unlike SHFM1, SHFM4 and SHFM5. G-banding and FISH failed to identify any chromosomal abnormalities. We also performed mutation screening by SSCP and DNA sequencing, as well as loss of heterozygosity (LOH) analysis, to exclude the possibility that SHFM4 or SHFM5 were involved; these revealed no mutations in gene p63 and no LOH on 2q31, respectively. It therefore appears that the Korean SHFM may be caused by mutation of SHFM3. In fact, linkage analysis using informative microsatellite markers indicated that SHFM3 was linked to D10S577 with a maximum LOD score of 1.15 at recombination fraction zero. Finally, we identified two novel alleles (191 and 211 bp) of D10S577 that have not been found in Western populations.

Dual matrilineal geographic distribution of Korean type 2 diabetes mellitus-associated -11,377 G adiponectin allele.

The present study was performed to identify the susceptible single nucleotide polymorphisms (SNPs) for the prediction of Korean type 2 diabetes mellitus (T2DM) and to clarify the matrilineal origin of Korean T2DM­specific SNPs. Fourteen SNPs from the adiponectin (ADIPOQ), hepatocyte nuclear factor 4α, phosphoenolpyruvate carboxykinase 1 and glucokinase genes in the Korean population were analyzed. Only one SNP, ­11,377 C/G on the ADIPOQ gene, was finally determined to be responsible for the incidence of Korean T2DM (P=0.028). The G­T­T­A haplotype at positions ­11,377, +45, +276 and +349 on the ADIPOQ gene was also associated with a high incidence of Korean T2DM (P=0.023). In addition, the susceptibility of Korean individuals to T2DM appears to be affected by their matrilineal origin. Of note, the group of Southern origin, consisting of mitochondrial DNA macrohaplogroups F and R, was predisposed to T2DM, whereas the group of Northern origin, consisting of haplogroups A and Y, was resistant to T2DM. This implied that the differential genetics between the two groups, which were formed from the initial peopling of the proto­Korean population via Southern and Northern routes to the present time, may explain their differing susceptibility to T2DM. In conclusion, from Southern Asia Northward, a matrilineal origin of Korean individuals appears to be responsible for the prevalence of Korean T2DM caused by the ­11,377 G allele.

Discoidin domain receptor 2 regulates the adhesion of fibroblasts to 3D collagen matrices.

The collagen matrix constitutes the primary extracellular matrix (ECM) portion of mammalian connective tissues in which the interaction of the cell and the surrounding collagen fibers has a significant impact on cell and tissue physiology, including morphogenesis, development and motility. Discoidin domain receptors (DDR1 and DDR2) have been identified as the receptor tyrosine kinases that are activated upon collagen binding. However, there is a lack of evidence regarding the effect of DDRs on the mechanical interaction between fibroblasts and ECM. In this study, we demonstrated that one of the major phosphotyrosine proteins in human fibroblasts during 3D collagen matrix polymerization is DDR2. Treatment of fibroblasts in 3D collagen matrices with platelet-derived growth factor (PDFG) has been shown to increase DDR2 phosphorylation. Silencing of DDR2 with siRNA in fibroblasts significantly reduced the number of dendritic extensions regardless of whether cells were cultured in the collagen or fibronectin 3D matrices. Decreasing dendritic extensions in DDR2-silenced cells has also been shown to decrease the ability of fibroblast entanglement to collagen fibrils in 3D collagen matrices. Finally, we also showed that the silencing of DDR2 decreased the cell migration in 3D nested collagen matrices but had no effect on 3D floating matrix contraction. Collectively, these results suggest that DDR2 functioning is required for the membrane dynamics to control the mechanical attachment of fibroblasts to the 3D collagen matrices in an integrin-independent manner.

Synthesis and biological evaluation of water-soluble organogermanium.

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.

Mitochondrial DNA analysis of ancient human bones excavated from Nukdo island, S.Korea.

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.

Identification of enteroviruses by using monoclonal antibodies against a putative common epitope.

A common epitope region of enteroviruses was identified by sequence-independent single-primer amplification (SISPA), followed by immunoscreening of 11 cDNA libraries from two Korean enterovirus isolates (echoviruses 7 and 30) and a coxsackievirus B3 (ATCC-VR 30). The putative common epitope region was localized in the N terminus of VP1 when the displayed recombinant proteins from the phages were chased by the convalescent-phase sera. The genomic region encoding the common epitope region was amplified and then expressed by using the vector pGEX-5X-1. The antigenicity of the expressed recombinant protein was identified by Western blotting with guinea pig antisera for six different serotypes of enteroviruses. After successive immunization of mice with the recombinant common epitope protein, splenocytes were extracted and hybridized with P3X63-Ag8-653 cells. A total of 24 hybridomas that produced monoclonal antibodies (MAbs) against the putative common epitope of enteroviruses were selected. Four of these were immunoglobulin G1 isotypes with a kappa light chain. These MAbs recognized 15 Korean endemic serotypes and prototypes of enteroviruses in an indirect immunofluorescence assay. These results suggest that the expressed protein might be a useful antigen for producing group common antibodies and that the use of the MAbs against the putative common epitope of enteroviruses might be a valuable diagnostic tool for rapidly identifying a broad range of enteroviruses.