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Mutagenesis Jun 2023The aim of the present study was to evaluate the compatibility of reconstructed 3D human small intestinal microtissues to perform the in vitro comet assay. The comet...
The aim of the present study was to evaluate the compatibility of reconstructed 3D human small intestinal microtissues to perform the in vitro comet assay. The comet assay is a common follow-up genotoxicity test to confirm or supplement other genotoxicity data. Technically, it can be performed utilizing a range of in vitro and in vivo assay systems. Here, we have developed a new reconstructed human intestinal comet (RICom) assay protocol for the assessment of orally ingested materials. The human intestine is a major site of food digestion and adsorption, first-pass metabolism as well as an early site of toxicant first contact and thus is a key site for evaluation. Reconstructed intestinal tissues were dosed with eight test chemicals: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), phenformin hydrochloride (Phen HCl), benzo[a]pyrene (BaP), 1,2-dimethylhydrazine hydrochloride (DMH), potassium bromate (KBr), glycidamide (GA), and etoposide (Etop) over a span of 48 h. The RICom assay correctly identified the genotoxicity of EMS, ENU, KBr, and GA. Phen HCl, a known non-genotoxin, did not induce DNA damage in the 3D reconstructed intestinal tissues whilst showing high cytotoxicity as assessed by the assay. The 3D reconstructed intestinal tissues possess sufficient metabolic competency for the successful detection of genotoxicity elicited by BaP, without the use of an exogenous metabolic system. In contrast, DMH, a chemical that requires liver metabolism to exert genotoxicity, did not induce detectable DNA damage in the 3D reconstructed intestinal tissue system. The genotoxicity of Etop, which is dependent on cellular proliferation, was also undetectable. These results suggest the RICom assay protocol is a promising tool for further investigation and safety assessment of novel ingested materials. We recommend that further work will broaden the scope of the 3D reconstructed intestinal tissue comet assay and facilitate broader analyses of genotoxic compounds having more varied modes of actions.
Topics: Humans; Comet Assay; DNA Damage; Mutagenicity Tests; Ethyl Methanesulfonate; Ethylnitrosourea; Intestines; Mutagens
PubMed: 37115513
DOI: 10.1093/mutage/gead011 -
ACS Omega Sep 2021Graphene-based drug carriers provide a promising addition to current cancer drug delivery options. Increased accessibility of high-quality graphene made by...
Graphene-based drug carriers provide a promising addition to current cancer drug delivery options. Increased accessibility of high-quality graphene made by plasma-enhanced chemical vapor deposition (PE-CVD) makes it an attractive material to revisit in comparison to the widely studied graphene oxide (GO) in drug delivery. Here, we show the potential of repurposing the metabolic drug phenformin for cancer treatment in terms of stability, binding, and pH-responsive release. Using covalent attachment of poly(ethylene glycol) (PEG) onto pristine (PE-CVD) graphene, we show that PEG stabilized graphene nanosheets (PGNS) are stable in aqueous solutions and exhibit higher binding affinity toward phenformin than GO. Moreover, we experimentally demonstrate an improved drug release from PGNS than GO at pH levels lower than physiological conditions, yet comparable to that found in tumor microenvironments.
PubMed: 34604644
DOI: 10.1021/acsomega.1c03283 -
Science Advances Nov 2020Mitochondria-derived reactive oxygen species (mROS) are required for the survival, proliferation, and metastasis of cancer cells. The mechanism by which mitochondrial...
Mitochondria-derived reactive oxygen species (mROS) are required for the survival, proliferation, and metastasis of cancer cells. The mechanism by which mitochondrial metabolism regulates mROS levels to support cancer cells is not fully understood. To address this, we conducted a metabolism-focused CRISPR-Cas9 genetic screen and uncovered that loss of genes encoding subunits of mitochondrial complex I was deleterious in the presence of the mitochondria-targeted antioxidant mito-vitamin E (MVE). Genetic or pharmacologic inhibition of mitochondrial complex I in combination with the mitochondria-targeted antioxidants, MVE or MitoTEMPO, induced a robust integrated stress response (ISR) and markedly diminished cell survival and proliferation in vitro. This was not observed following inhibition of mitochondrial complex III. Administration of MitoTEMPO in combination with the mitochondrial complex I inhibitor phenformin decreased the leukemic burden in a mouse model of T cell acute lymphoblastic leukemia. Thus, mitochondrial complex I is a dominant metabolic determinant of mROS-dependent cellular fitness.
PubMed: 33148642
DOI: 10.1126/sciadv.abb7272 -
Cancers May 2020Lung adenocarcinoma cells express high levels of , an enzyme of the one-carbon pathway that catalyzes the conversion of 10-formyltetrahydrofolate into tetrahydrofolate...
Lung adenocarcinoma cells express high levels of , an enzyme of the one-carbon pathway that catalyzes the conversion of 10-formyltetrahydrofolate into tetrahydrofolate and NAD(P)H. In this study, we evaluated the potential of as a therapeutic target by deleting the gene in mice, a model of spontaneous non-small cell lung cancer (NSCLC). Reporter assays revealed KRAS-mediated upregulation of the promoter in human NSCLC cells. mice exhibited a normal phenotype, with a 10% decrease in driven lung tumorigenesis. By contrast, the inhibition of oxidative phosphorylation inhibition using phenformin in ; mice dramatically decreased the number of tumor nodules and tumor area by up to 50%. Furthermore, combined treatment with pan-ALDH inhibitor and phenformin showed a decreased number and area of lung tumors by 70% in the lung cancer model. Consistent with this, previous work showed that the combination of knockdown and phenformin treatment decreased ATP production by as much as 70% in NSCLS cell lines. Taken together, these results suggest that the combined inhibition of ALDH activity and oxidative phosphorylation represents a promising therapeutic strategy for NSCLC.
PubMed: 32481524
DOI: 10.3390/cancers12061382 -
Communications Biology Jan 2023Years of use of the antidiabetic drug metformin has long been associated with the risk of vitamin B12 (B12) deficiency in type 2 diabetes (T2D) patients, although the...
Years of use of the antidiabetic drug metformin has long been associated with the risk of vitamin B12 (B12) deficiency in type 2 diabetes (T2D) patients, although the underlying mechanisms are unclear. Accumulating evidence has shown that metformin may exert beneficial effects by altering the metabolism of the gut microbiota, but whether it induces human B12 deficiency via modulation of bacterial activity remains poorly understood. Here, we show that both metformin and the other biguanide drug phenformin markedly elevate the accumulation of B12 in E. coli. By functional and genomic analysis, we demonstrate that both biguanides can significantly increase the expression of B12 transporter genes, and depletions of vital ones, such as tonB, nearly completely abolish the drugs' effect on bacterial B12 accumulation. Via high-throughput screens in E. coli and C. elegans, we reveal that the TetR-type transcription factor RcdA is required for biguanide-mediated promotion of B12 accumulation and the expressions of B12 transporter genes in bacteria. Together, our study unveils that the antidiabetic drug metformin helps bacteria gather B12 from the environment by increasing the expressions of B12 transporter genes in an RcdA-dependent manner, which may theoretically reduce the B12 supply to T2D patients taking the drug over time.
Topics: Humans; Animals; Hypoglycemic Agents; Metformin; Diabetes Mellitus, Type 2; Vitamin B 12; Caenorhabditis elegans; Escherichia coli; Vitamin B 12 Deficiency
PubMed: 36693976
DOI: 10.1038/s42003-023-04475-0 -
Frontiers in Bioscience (Landmark... Aug 2023Activation of the unfolded protein response (UPR) is closely related to the pathogenesis of many metabolic disorders. Accumulating evidence also shows that UPR and...
BACKGROUND
Activation of the unfolded protein response (UPR) is closely related to the pathogenesis of many metabolic disorders. Accumulating evidence also shows that UPR and metabolic signaling pathways are interdependent. The AMP-activated protein kinase (AMPK) signal pathway controls the energy balance of eukaryotes. The aim of this study was therefore to investigate the possible interaction between AMPK signaling and UPR in muscle cells exposed to saturated fatty acids, as well as the potential mechanism.
METHODS
The saturated fatty acid palmitate was used to induce UPR in C2C12 myotubes. Compound C or knockdown of AMPKα with short hairpin RNA (shRNA) were used to inhibit the AMPK signaling pathway in palmitate-treated muscle cells. AMPK signaling in myotubes was activated using 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICAR) or ex229. C2C12 myotubes were pre-treated with taurourdodeoxycholic acid (TUDCA) to inhibit UPR before adding palmitate. Real-time PCR and Western blotting were performed to evaluate the expression of UPR markers and activation of AMPK.
RESULTS
Palmitate treatment induced UPR in C2C12 myotubes while activating AMPK signaling. Inhibition of the AMPK pathway with compound C or AMPK shRNA reduced palmitate-induced activation of UPR, while inhibition of UPR with TUDCA reduced palmitate-induced AMPK activation. This indicates a positive feedback loop between UPR and AMPK. Furthermore, activation of the AMPK pathway with AICAR or ex229 caused a dose-dependent upregulation of UPR markers, including activating transcription factor 4 (ATF4), binding immunoglobulin protein (BIP), and growth arrest and DNA damage-inducible 34 (GADD34) protein.
CONCLUSIONS
These results provide the first evidence that AMPK signaling is involved in the early activation of UPR caused by saturated fatty acids in skeletal muscle. Furthermore, they indicate that physiological or pharmacological activation of the AMPK pathway (e.g., by exercise or phenformin, respectively) can promote muscle health and function, thereby improving the quality of life in individuals with metabolic disorders due to a high-fat diet or obesity.
Topics: Humans; AMP-Activated Protein Kinases; Feedback; Quality of Life; Muscle Cells
PubMed: 37664919
DOI: 10.31083/j.fbl2808159 -
Scientific Reports Jan 2024Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer. Since the tricarboxylic acid cycle is widely involved in tumor metabolic reprogramming and...
Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer. Since the tricarboxylic acid cycle is widely involved in tumor metabolic reprogramming and cuproptosis, investigating related genes may help to identify prognostic signature of patients with HCC. Data on patients with HCC were sourced from public datasets, and were divided into train, test, and single-cell cohorts. A variety of machine learning algorithms were used to identify different molecular subtypes and determine the prognostic risk model. Our findings revealed that the risk score (TRscore), based on the genes OGDHL, CFHR4, and SPP1, showed excellent predictive performance in different datasets. Pathways related to cell cycle and immune inflammation were enriched in the high-risk group, whereas metabolism-related pathways were significantly enriched in the low-risk group. The high-risk group was associated with a greater number of mutations of detrimental biological behavior and higher levels of immune infiltration, immune checkpoint expression, and anti-cancer immunotherapy response. Low-risk patients demonstrated greater sensitivity to erlotinib and phenformin. SPP1 was mainly involved in the interaction among tumor-associated macrophages, T cells, and malignant cells via SPP1-CD44 and SPP1-(ITGA5 + ITGB1) ligand-receptor pairs. In summary, our study established a prognostic model, which may contribute to individualized treatment and clinical management of patients with HCC.
Topics: Humans; Carcinoma, Hepatocellular; Prognosis; Citric Acid Cycle; Liver Neoplasms; Algorithms; Tumor Microenvironment
PubMed: 38282028
DOI: 10.1038/s41598-024-52632-0 -
Journal of Nanobiotechnology Nov 2021Mild-temperature photothermal therapy (mild-PTT) has emerged as a highly promising antitumor strategy by triggering immunogenic cell death (ICD) to elicit both innate...
Selectively down-regulated PD-L1 by albumin-phenformin nanoparticles mediated mitochondrial dysfunction to stimulate tumor-specific immunological response for enhanced mild-temperature photothermal efficacy.
BACKGROUND
Mild-temperature photothermal therapy (mild-PTT) has emerged as a highly promising antitumor strategy by triggering immunogenic cell death (ICD) to elicit both innate and adaptive immune responses for tumor control. However, mild-PTT still leads to the risk of tumor recurrence or metastasis because it could hardly completely eradicate tumors due to its impaired immunological efficacy owing to the enhanced PD-L1 expression in tumor cells after treatment.
RESULTS
In this study, we described a hydrogen peroxide (HO) responsive manganese dioxide mineralized albumin nanocomposite loading with mitochondria function inhibitor phenformin (PM) and near-infrared photothermal dye indocyanine green (ICG) by modified two-step biomineralization method. In combination with ICG induced mild-PTT and PM mediated mitochondria dysfunction, PD-L1 expression was obviously down-regulated and the generated immunological responses was able to effectively attack the remaining tumor cells. Meanwhile, the risk of tumor metastasis was effectively inhibited by reducing the expression of tumor invasion-related signal molecules (TGF-β and vimentin) after combining treatment.
CONCLUSION
Such a strategy offers novel insight into the development of nanomedicine for mild-PTT as well as cancer immunotherapy, which can provide protection against tumor relapse post elimination of their initial and metastatic tumors.
Topics: Albumins; Animals; Antineoplastic Agents; B7-H1 Antigen; Biomineralization; Cell Line, Tumor; Down-Regulation; Hydrogen Peroxide; Indocyanine Green; Manganese Compounds; Mice; Mitochondria; Nanoparticles; Oxides; Phenformin; Photothermal Therapy
PubMed: 34794446
DOI: 10.1186/s12951-021-01124-8 -
International Journal of Molecular... Feb 2020Melanin in the epidermis is known to ultimately regulate human skin pigmentation. Recently, we exploited a phenotypic-based screening system composed of ex vivo human...
Melanin in the epidermis is known to ultimately regulate human skin pigmentation. Recently, we exploited a phenotypic-based screening system composed of ex vivo human skin cultures to search for effective materials to regulate skin pigmentation. Since a previous study reported the potent inhibitory effect of metformin on melanogenesis, we evaluated several biguanide compounds. The unexpected effect of phenformin, once used as an oral anti-diabetic drug, on cutaneous darkening motivated us to investigate its underlying mechanism utilizing a chemical genetics approach, and especially to identify alternatives to phenformin because of its risk of severe lactic acidosis. Chemical pull-down assays with phenformin-immobilized beads were performed on lysates of human epidermal keratinocytes, and subsequent mass spectrometry identified 7-dehydrocholesterol reductase (DHCR7). Consistent with this, AY9944, an inhibitor of DHCR7, was found to decrease autophagic melanosome degradation in keratinocytes and to intensely darken skin in ex vivo cultures, suggesting the involvement of cholesterol biosynthesis in the metabolism of melanosomes. Thus, our results validated the combined utilization of the phenotypic screening system and chemical genetics as a new approach to develop promising materials for brightening/lightening and/or tanning technologies.
Topics: Cholesterol; Female; Humans; Keratinocytes; Male; Melanocytes; Melanosomes; Oxidoreductases Acting on CH-CH Group Donors; Phenformin; Skin Pigmentation; trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride
PubMed: 32093380
DOI: 10.3390/ijms21041451 -
BMC Cancer Jan 2024Pancreatic adenocarcinoma (PDAC) ranks as the fourth leading cause for cancer-related deaths worldwide. N6-methyladenosine (m6A) and long non-coding RNAs (lncRNAs) are...
BACKGROUND
Pancreatic adenocarcinoma (PDAC) ranks as the fourth leading cause for cancer-related deaths worldwide. N6-methyladenosine (m6A) and long non-coding RNAs (lncRNAs) are closely related with poor prognosis and immunotherapeutic effect in PDAC. The aim of this study is to construct and validate a m6A-related lncRNAs signature and assess immunotherapeutic drug sensitivity in PDAC.
METHODS
RNA-seq data for 178 cases of PDAC patients and 167 cases of normal pancreatic tissue were obtained from TCGA and GTEx databases, respectively. A set of 21 m6A-related genes were downloaded based on the previous report. Co-expression network was conducted to identify m6A-related lncRNAs in PDAC. Cox analyses and least absolute shrinkage and selection operator (Lasso) regression model were used to construct a risk prognosis model. The relationship between signature genes and immune function was explored by single-sample GSEA (ssGSEA). The tumor immune dysfunction and exclusion (TIDE) score and tumor mutation burden (TMB) were utilized to evaluate the response to immunotherapy. Furthermore, the expression levels of 4 m6A-related lncRNAs on PDAC cell lines were measured by the quantitative real-time PCR (qPCR). The drug sensitivity between the high- and low-risk groups was validated using PDAC cell lines by Cell-Counting Kit 8 (CCK8).
RESULTS
The risk prognosis model was successfully constructed based on 4 m6A-related lncRNAs, and PDAC patients were divided into the high- and low-risk groups. The overall survival (OS) of the high-risk groups was more unfavorable compared with the low-risk groups. Receiver operating characteristic (ROC) curves demonstrated that the risk prognosis model reasonably predicted the 2-, 3- and 5-year OS of PDAC patients. qPCR analysis confirmed the decreased expression levels of 4 m6A-related lncRNAs in PDAC cells compared to the normal pancreatic cells. Furthermore, CCK8 assay revealed that Phenformin exhibited higher sensitivity in the high-risk groups, while Pyrimethamine exhibited higher sensitivity in the low-risk groups.
CONCLUSION
The prognosis of patients with PDAC were well predicted in the risk prognosis model based on m6A-related lncRNAs, and selected immunotherapy drugs have potential values for the treatment of pancreatic cancer.
Topics: Humans; Adenocarcinoma; RNA, Long Noncoding; Pancreatic Neoplasms; Pancreas; Adenine
PubMed: 38262966
DOI: 10.1186/s12885-024-11885-8