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Journal of Cachexia, Sarcopenia and... Dec 2021Chemotherapy is an essential treatment to combat solid tumours and mitigate metastasis. Chemotherapy causes side effects including muscle wasting and weakness. Regulated...
BACKGROUND
Chemotherapy is an essential treatment to combat solid tumours and mitigate metastasis. Chemotherapy causes side effects including muscle wasting and weakness. Regulated in Development and DNA Damage Response 1 (REDD1) is a stress-response protein that represses the mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1), and its expression is increased in models of muscle wasting. The aim of this study was to determine if deletion of REDD1 is sufficient to attenuate chemotherapy-induced muscle wasting and weakness in mice.
METHODS
C2C12 myotubes were treated with carboplatin, and changes in myotube diameter were measured. Protein synthesis was measured by puromycin incorporation, and REDD1 mRNA and protein expression were analysed in myotubes treated with carboplatin. Markers of mTORC1 signalling were measured by western blot. REDD1 global knockout mice and wild-type mice were treated with a single dose of carboplatin and euthanized 7 days later. Body weight, hindlimb muscle weights, forelimb grip strength, and extensor digitorum longus whole muscle contractility were measured in all groups. Thirty minutes prior to euthanasia, mice were injected with puromycin to measure puromycin incorporation in skeletal muscle.
RESULTS
C2C12 myotube diameter was decreased at 24 (P = 0.0002) and 48 h (P < 0.0001) after carboplatin treatment. Puromycin incorporation was decreased in myotubes treated with carboplatin for 24 (P = 0.0068) and 48 h (P = 0.0008). REDD1 mRNA and protein expression were increased with carboplatin treatment (P = 0.0267 and P = 0.0015, respectively), and this was accompanied by decreased phosphorylation of Akt T (P < 0.0001) and S (P = 0.0006), p70S6K T (P = 0.0002), and 4E-binding protein 1 S (P = 0.0341), all markers of mTORC1 activity. REDD1 mRNA expression was increased in muscles from mice treated with carboplatin (P = 0.0295). Loss of REDD1 reduced carboplatin-induced body weight loss (P = 0.0013) and prevented muscle atrophy in mice. REDD1 deletion prevented carboplatin-induced decrease of protein synthesis (P = 0.7626) and prevented muscle weakness.
CONCLUSIONS
Carboplatin caused loss of body weight, muscle atrophy, muscle weakness, and inhibition of protein synthesis. Loss of REDD1 attenuates muscle atrophy and weakness in mice treated with carboplatin. Our study illustrates the importance of REDD1 in the regulation of muscle mass with chemotherapy treatment and may be an attractive therapeutic target to combat cachexia.
Topics: Animals; Antineoplastic Agents; Mice; Mice, Knockout; Muscle, Skeletal; Muscular Atrophy; Transcription Factors
PubMed: 34664403
DOI: 10.1002/jcsm.12795 -
FASEB Journal : Official Publication of... Apr 2021Skeletal muscle is the largest organ of the body, the development of skeletal muscle is very important for the health of the animal body. Prolyl hydroxylases (PHDs) are...
Skeletal muscle is the largest organ of the body, the development of skeletal muscle is very important for the health of the animal body. Prolyl hydroxylases (PHDs) are the classical regulator of the hypoxia inducible factor (HIF) signal pathway, many researchers found that PHDs are involved in the muscle fiber type transformation, muscle regeneration, and myocyte differentiation. However, whether PHDs can impact the protein turnover of skeletal muscle is poorly understood. In this study, we constructed denervated muscle atrophy mouse model and found PHD3 was highly expressed in the atrophic muscles and there was a significant correlation between the expression level of PHD3 and skeletal muscle weight which was distinct from PHD1 and PHD2. Then, the similar results were getting from the different weight muscles of normal mice. To further verify the relationship between PHD3 and skeletal muscle protein turnover, we established a PHD3 interference model by injecting PHD3 sgRNA virus into tibialis anterior muscle (TA) muscle of MCK-Cre-cas9 mice and transfecting PHD3 shRNA lentivirus into primary satellite cells. It was found that the Knock-out of PHD3 in vivo led to a significant increase in muscle weight and muscle fiber area (P < .05). Besides, the activity of protein synthesis signal pathway increased significantly, while the protein degradation pathway was inhibited evidently (P < .05). In vitro, the results of 5-ethynyl-2'-deoxyuridine (EdU) and tetramethylrhodamine ethyl ester (TMRE) fluorescence detection showed that PHD3 interference could lead to a decrease in cell proliferation and an increase of cell apoptosis. After the differentiation of satellite cells, the production of puromycin in the interference group was higher than that in the control group, and the content of 3-methylhistidine in the interference group was lower than that in the control group (P < .05) which is consistent with the change of protein turnover signal pathway in the cell. Mechanistically, there is an interaction between PHD3, NF-κB, and IKBα which was detected by immunoprecipitation. With the interfering of PHD3, the expression of the inflammatory signal pathway also significantly decreased (P < .05). These results suggest that PHD3 may affect protein turnover in muscle tissue by mediating inflammatory signal pathway. Finally, we knocked out PHD3 in denervated muscle atrophy mice and LPS-induced myotubes atrophy model. Then, we found that the decrease of PHD3 protein level could alleviate the muscle weight and muscle fiber reduction induced by denervation in mice. Meanwhile, the protein level of the inflammatory signal pathway and the content of 3-methylhistidine in denervated atrophic muscle were also significantly reduced (P < .05). In vitro, PHD3 knock-out could alleviate the decrease of myotube diameter induced by LPS, and the expression of protein synthesis pathway was also significantly increased (P < .05). On the contrary, the expression level of protein degradation and inflammatory signal pathway was significantly decreased (P < .05). Through these series of studies, we found that the increased expression of PHD3 in denervated muscle might be an important regulator in inducing muscle atrophy, and this process is likely to be mediated by the inflammatory NF-κB signal pathway.
Topics: Animals; Denervation; Gene Expression Regulation; Hypertrophy; Inflammation; Methylhistidines; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Muscular Atrophy; NF-kappa B; Procollagen-Proline Dioxygenase; Puromycin; Satellite Cells, Skeletal Muscle; Signal Transduction
PubMed: 33749901
DOI: 10.1096/fj.202002049R -
Scientific Reports Sep 2022Leucine (Leu) regulates protein synthesis and degradation via activation of mammalian target of rapamycin complex 1 (mTORC1). Glutamine (Gln) synergistically promotes...
Leucine (Leu) regulates protein synthesis and degradation via activation of mammalian target of rapamycin complex 1 (mTORC1). Glutamine (Gln) synergistically promotes mTORC1 activation with Leu via glutaminolysis and Leu absorption via an antiporter. However, Gln has also been shown to inhibit mTORC1 activity. To resolve this paradox, we aimed to elucidate the effects of Gln on Leu-mediated mTORC1 activation. We administered Leu, Gln, tryptophan, Leu + Gln, or Leu + tryptophan to mice after 24-h fasting. The mice were then administered puromycin to evaluate protein synthesis and the gastrocnemius muscle was harvested 30 min later. Phosphorylated eukaryotic initiation factor 4E-binding protein 1, 70-kDa ribosomal protein S6 kinase 1, and Unc-51 like kinase 1 levels were the highest in the Leu + Gln group and significantly increased compared with those in the control group; however, Gln alone did not increase the levels of phosphorylated proteins. No difference in glutamate dehydrogenase activity was observed between the groups. Leu concentrations in the gastrocnemius muscle were similar in the Leu-intake groups. Our study highlights a novel mechanism underlying the promotive effect of Gln on Leu-mediated mTORC1 activation, providing insights into the pathway through which amino acids regulate muscle protein metabolism.
Topics: Amino Acids; Animals; Antiporters; Eating; Eukaryotic Initiation Factor-4E; Glutamate Dehydrogenase; Glutamine; Leucine; Mechanistic Target of Rapamycin Complex 1; Mice; Muscle Proteins; Phosphorylation; Puromycin; Tryptophan
PubMed: 36151270
DOI: 10.1038/s41598-022-20251-2 -
Biochemical and Biophysical Research... Jun 2021Klotho deficiency was observed in virtually all kinds of kidney disease and is thought to play a critical role in podocyte injury. However, the underline mechanisms...
Klotho deficiency was observed in virtually all kinds of kidney disease and is thought to play a critical role in podocyte injury. However, the underline mechanisms involved in podocyte injury remain unknown. miRNAs have diverse regulatory roles, and miR-30 family members were essential for podocyte homeostasis. Our study revealed that Klotho and miR-30s were downregulated in PAN-treated podocytes. The ectopic expression of Klotho ameliorates PAN induced podocyte apoptosis through upregulating miR-30a and downregulating Ppp3ca, Ppp3cb, Ppp3r1, and Nfact3 expression, which are the known targets of miR-30s. We also found that Klotho regulates TRPC6 via miR-30a to activate calcium/calcineurin signaling. Further, glucocorticoid (Dexamethasone, DEX) was found to sustain Klotho and miR-30a levels during PAN treatment in vitro. Eventually, in rats, PAN treatment substantially downregulated Klotho and miR-30a levels, lead to podocyte injury and increased proteinuria. The transfer of exogenous Klotho to podocytes of PAN-treated rats could increase miR-30a expression, reduce TRPC6 expression, and also ameliorated podocyte injury and proteinuria. In conclusion, Klotho, acting on miR-30s, which directly regulates its target genes, contributes to podocyte apoptosis induced by PAN. It is a novel mechanism underlying PAN-induced podocyte injury.
Topics: Animals; Calcium Signaling; Cells, Cultured; Dexamethasone; Disease Models, Animal; Down-Regulation; Glucuronidase; Humans; Kidney Diseases; Klotho Proteins; Male; MicroRNAs; Podocytes; Puromycin Aminonucleoside; Rats; Rats, Wistar; TRPC6 Cation Channel
PubMed: 33862465
DOI: 10.1016/j.bbrc.2021.04.003 -
3 Biotech Aug 2021A novel aminopeptidase B (APB-AN) was identified from CGMCC 3.1454 for the first time and was cloned and expressed in . The mature enzyme of approximately 100 kDa was...
A novel aminopeptidase B (APB-AN) was identified from CGMCC 3.1454 for the first time and was cloned and expressed in . The mature enzyme of approximately 100 kDa was purified for characterization. The optimum pH and temperature of the recombinant APB-AN were determined to be 7.0 and 40 °C, respectively. The enzyme was stable below 40 °C and at pH values from 5.0 to 8.0. The and values were determined to be 0.61 mmol/L and 11.45 mmol/L/min, respectively, using Arg-NA as the substrate. APB-AN was inhibited by Cu and Fe and activated by Co and Na. Most metal chelators (Ca, Mg and Mn) and aminopeptidase inhibitors (bestatin and puromycin) suppressed its activity. APB-AN was found to be active towards 13 kinds of amino acid -nitroanilide (NA) substrates:Arg-NA, Lys-NA, Tyr- pNA, Trp-NA, Phe-NA, His-NA, Ala-NA, Met-NA, Leu-NA, Glu-NA, Val-NA, Pro-NA and Ile-NA, and the most preferred N-terminal amino acids were arginine and lysine. APB-AN also hydrolyzed 4 natural proteins: casein, bovine serum albumin, soy protein isolate and water-soluble wheat protein. It is expected that APB-AN has potential food processing applications.
PubMed: 34290949
DOI: 10.1007/s13205-021-02915-4 -
Life Sciences Oct 2022Drug-induced nephrotoxicity is frequently reported. However, the mechanisms underlying nephrotoxic medications and their overlapping molecular events, which might have...
Drug-induced nephrotoxicity is frequently reported. However, the mechanisms underlying nephrotoxic medications and their overlapping molecular events, which might have therapeutic value, are unclear. We performed a genome-wide analysis of gene expression and a gene set enrichment analysis to identify common and unique pathways associated with the toxicity of colistin, ifosfamide, indomethacin, and puromycin. Rats were randomly allocated into the treatment or control group. The treatment group received a toxic dose once daily of each investigated drug for 1 week. Differentially expressed genes were found in the drug-treated kidney and liver compared to the control, except for colistin in the liver. Upregulated pathways were mainly related to cell death, cell cycle, protein synthesis, and immune response modulation in the kidney. Cell cycle was upregulated by all drugs. Downregulated pathways were associated with carbon metabolism, amino acid metabolism, and fatty acid metabolism. Indomethacin, colistin, and puromycin shared the most altered pathways in the kidney. Ifosfamide and indomethacin affected molecular processes greatly in the liver. Our findings provide insight into the mechanisms underlying the renal and hepatic adverse effects of the four drugs. Further investigation should explore the combinatory drug therapies that attenuate the toxic effects and maximize the effectiveness of nephrotoxic drugs.
Topics: Animals; Colistin; Gene Expression; Ifosfamide; Indomethacin; Kidney; Puromycin; Rats
PubMed: 35850247
DOI: 10.1016/j.lfs.2022.120801 -
Seminars in Nuclear Medicine Sep 2023It is important to constantly monitor developments in the preclinical imaging arena of infection. Firstly, novel radiopharmaceuticals with the correct characteristics... (Review)
Review
It is important to constantly monitor developments in the preclinical imaging arena of infection. Firstly, novel radiopharmaceuticals with the correct characteristics must be identified to funnel into the clinic. Secondly, it must be evaluated if enough innovative research is being done and adequate resources are geared towards the development of radiopharmaceuticals that could feed into the Nuclear Medicine Clinic in the near future. It is proposed that the ideal infection imaging agent will involve PET combined with CT but more ideally MRI. The radiopharmaceuticals currently presented in preclinical literature have a wide selection of vectors and targets. Ionic formulations of PET-radionuclides such CuCl and GaCl are evaluated for bacterial infection imaging. Many small molecule based radiopharmaceuticals are being investigated with the most prominent targets being cell wall synthesis, maltodextrin transport (such as [F]F-maltotriose), siderophores (bacterial and fungal infections), the folate synthesis pathway (such as [F]F-PABA) and protein synthesis (radiolabelled puromycin). Mycobacterial specific antibiotics, antifungals and antiviral agents are also under investigation as infection imaging agents. Peptide based radiopharmaceuticals are developed for bacterial, fungal and viral infections. The radiopharmaceutical development could even react quickly enough on a pandemic to develop a SARS-CoV-2 imaging agent in a timely fashion ([Cu]Cu-NOTA-EK1). New immuno-PET agents for the imaging of viruses have recently been published, specifically for HIV persistence but also for SARS-CoV2. A very promising antifungal immuno-PET agent (hJ5F) is also considered. Future technologies could include the application of aptamers and bacteriophages and even going as far as the design of theranostic infection. Another possibility would be the application of nanobodies for immuno-PET applications. Standardization and optimization of the preclinical evaluation of radiopharmaceuticals could enhance clinical translation and reduce time spent in pursuing less than optimal candidates.
Topics: Humans; Radiopharmaceuticals; RNA, Viral; COVID-19; SARS-CoV-2; Positron-Emission Tomography
PubMed: 37012169
DOI: 10.1053/j.semnuclmed.2023.03.001 -
Molecular Biotechnology Aug 2023We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in...
We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in chronic myeloid leukemia (CML). MiR-217 overexpression and the knock-down lentiviral vector with puromycin resistance were constructed and packaged within recombinant lentivirus. Stably transfected K562 cells were obtained through puromycin screening, and the qPCR assay detected the relative expression of the target gene. The proliferation, apoptosis, and methylation level of PER2 within cultured cells were detected using the CCK-8 assay, flow cytometry, and TaqMan real‑time fluorescence quantitative methylation-specific PCR. qPCR and Western blot detected the expression of miR-217-related genes within the constructed K562 cell model. Colony PCR and sequencing proved that recombinant lentivirus expression vectors pSE16 and pSE17 were correctly constructed. The lentivirus titer was 2.95 × 10 and 2.61 × 10 IU/mL. The miR-217 expression level was high in pSE5316-K562 cells, and that of the miR-217 sponge was high in pSE5317-K562 cells. Overexpressed miR-217 could inhibit the K562 cell proliferation and induce apoptosis. Inhibition of miR-217 enhanced the expression of DNMT3A, decreased the PER2 expression, and elevated the degree of PER2 methylation. The miR-217 overexpression and knock-down of the K562 cell line were successfully constructed, providing a tool for further exploring the miR-217 mechanism in CML. DNMT3A could be the molecular target of miR-217 by regulating PER2 gene methylation and getting involved with the occurrence and development of CML.
Topics: Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; K562 Cells; Lentivirus; Cells, Cultured; Apoptosis; Cell Proliferation; MicroRNAs; Cell Line, Tumor
PubMed: 36495416
DOI: 10.1007/s12033-022-00615-9 -
Methods in Molecular Biology (Clifton,... 2022High-throughput CRISPR guide RNA (gRNA) library screen, that is, CRISPR/Cas9 screen, enables the unbiased identification of gene functions in a variety of biological...
High-throughput CRISPR guide RNA (gRNA) library screen, that is, CRISPR/Cas9 screen, enables the unbiased identification of gene functions in a variety of biological processes. Typical pooled CRISPR/Cas9 screen couples a gRNA library and a guided Cas9 or dCas9 endonuclease to target specific gene loci, and then systematically uncover the causal link between candidate genes and observed cellular phenotypes via gRNA depletion or enrichment in screens. Here, we describe a detailed method of puromycin (PURO) concentration titration and lentiviral CRISPR gRNA library titration in Cas9 expressing monoclonal human iPSC line (Cas9+MNhiPSC) prior to performing the screens, conducting pooled CRISPR gRNA library screens in Cas9+MNhiPSC, genomic DNA extraction from the selected cell subpopulation and sequencing library preparation as well as next generation sequencing (NGS) to generate gRNA read counts. In CRISPR/Cas9 screen, we aim for 30% transduction efficiency (i.e., multiplicity of infection = 0.3) to ensure most of infected cells receive only one gRNA. The principles in this method can be applied to CRISPR perturbation (knockout, activation, repression or base editing) screens with other CRISPR gRNA libraries across many other cell models and other species.
Topics: CRISPR-Cas Systems; Endonucleases; Gene Library; Humans; Induced Pluripotent Stem Cells; RNA, Guide, CRISPR-Cas Systems
PubMed: 35347694
DOI: 10.1007/7651_2021_455 -
Asian Pacific Journal of Cancer... May 2022Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also...
UNLABELLED
Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also toxic for normal cell, not specific to the target cells. Consequently, chemotherapy has various side effects. Monoclonal antibody (MAb) has been developed for specific therapy which only has killing effect in cancer cells, but the survival rate of most MAbs around 20%. Therefore, in clinical practice, MAbs administration should combine with chemotherapeutic agents. For effectiveness of therapy and to minimalize adverse effects, anticancer agent with selective cytotoxic effect on target cells is needed, the immunotoxin.
OBJECTIVE
This study introduces a novel approach to conjugate monoclonal antibody (Cetuximab) and toxin (Puromycin), in order to selectively inhibit proliferation of triple negative breast cancer (TNBC) and to enhance the efficacy of MAb in target cells killing.
METHODS
Cetuximab was conjugated with Puromycin using a linker, i.e SATP (Succinimidyl-acetylthiopropionate) and tested on triple negative breast cancer cell lines (MDA-MB-231) which expressed EGFR (epidermal growth factor receptor). Cetuximab is MAb which targets EGFR. MCF-7 was used as control cells since it has low or no EGFR expression. Cell counting were conducted as viability assay at 24 hours, 48 hours, and 72 hours after treatment.
RESULTS
The results showed significant reduction of live cells number in Conjugate 20 µg/mL cultured in MDA-MB-231 compared to MCF-7 after 24 hours, 48 hours, and 72 hours incubation. In all time period of incubation, significant reduction of MDA-MB-231 live cells number was also observed in Conjugate 20 µg/mL compared to Cetuximab 20 µg/mL.
CONCLUSION
Synthesized conjugate showed its target-specific effect in TNBC and improved the efficacy of Cetuximab on TNBC. In the future, this conjugate can be a potential anticancer therapy in treating triple-negative breast cancer.
Topics: Antibodies, Monoclonal; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cetuximab; ErbB Receptors; Humans; Puromycin; Triple Negative Breast Neoplasms
PubMed: 35633567
DOI: 10.31557/APJCP.2022.23.5.1803