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Reproductive Biology and Endocrinology... Feb 2019Infertile men have higher levels of semen reactive oxygen species (ROS) than fertile men. High levels of semen ROS can cause sperm dysfunction, sperm DNA damage and... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
Infertile men have higher levels of semen reactive oxygen species (ROS) than fertile men. High levels of semen ROS can cause sperm dysfunction, sperm DNA damage and reduced male reproductive potential. This study investigated the effects of supplementation with N-acetyl-cysteine (NAC) on the sperm quality, chromatin integrity and levels of oxidative stress in infertile men.
METHODS
The study was carried out in the unit of ACECR Infertility Research Center, Qom, Iran. The patients consisted of 50 infertile men with asthenoteratozoospermia who received NAC (600 mg/d) orally for 3 months, after which they were compared with pre-treatment status. Semen was analyzed according to WHO (2010), followed by the assessment of protamine content [chromomycin A3 (CMA3)] and DNA integrity [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)]. Oxidative stress markers, i.e. total antioxidant capacity (TAC) and malondialdehyde (MDA), as well as hormonal profile (LH, FSH, Testosterone and Prolactin) were determined by ELISA kit.
RESULTS
After NAC treatment, patients' sperm count and motility increased significantly whereas abnormal morphology, DNA fragmentation and protamine deficiency showed significant decreases compared to pre-treatment levels (P < 0.05). Hormonal profile improvement was associated with lowered FSH and LH levels and increased amount of testosterone (P < 0.05). TAC significantly increased and MDA decreased with an inverse significant correlation between TAC and MDA (P < 0.05).
CONCLUSION
NAC oral supplementation may improve sperm parameters and oxidative/antioxidant status in infertile males.
Topics: Acetylcysteine; Adult; Chromatin; DNA Damage; Dietary Supplements; Free Radical Scavengers; Humans; Infertility, Male; Iran; Male; Oxidative Stress; Reactive Oxygen Species; Semen Analysis; Sperm Motility; Spermatozoa
PubMed: 30771790
DOI: 10.1186/s12958-019-0468-9 -
Frontiers in Immunology 2022Some first-line cytotoxic chemotherapics, doxorubicin, paclitaxel and oxaliplatin, induce activation of the immune system through immunogenic cell death (ICD). Tumor...
PURPOSE
Some first-line cytotoxic chemotherapics, doxorubicin, paclitaxel and oxaliplatin, induce activation of the immune system through immunogenic cell death (ICD). Tumor cells undergoing ICD function as a vaccine, releasing damage-associated molecular patterns (DAMPs), which act as adjuvants, and neoantigens of the tumor are recognized as antigens. ICD induction is rare, however it yields better and long-lasting antitumor responses to chemotherapy. Advanced metastatic melanoma (AMM) is incurable for more than half of patients. The discovery of ICD inducers against AMM is an interesting drug discovery strategy with high translational potential. Here we evaluated ICD induction of four highly cytotoxic chromomycins A (CA).
METHODS
ICD features and DAMPs were evaluated using several techniques with metastatic melanoma cell line (B16-F10) exposed to chromomcins A such as flow cytometry, western blot, RT-PCR and luminescence. Additionally vaccination assays with CA-treated cells in a syngeneic murine model (C57Bl/6) were performed to confirm ICD evaluating the immune cells activation and their antitumor activity.
RESULTS
B16-F10 treated with CA and doxorubicin exhibited ICD features such as autophagy and apoptosis, externalization of calreticulin, and releasing of HMGB1. However, CA-treated cells had the best profile, also inducing ATP release, ERp57 externalization, phosphorylation of eIF2α and altering expression of transcription of genes related to autophagy, endoplasmic reticulum stress, and apoptosis. Bona fide ICD induction by CA was confirmed by vaccination of C57BL/6 mice with CA-treated cells which activated antigen-presenting cells and T lymphocytes and stimulated antitumor activity.
CONCLUSION
CA induces bona fide immunogenic cell death on melanoma.
Topics: Mice; Animals; Immunogenic Cell Death; Cell Line, Tumor; Mice, Inbred C57BL; Antineoplastic Agents; Melanoma; Doxorubicin; Alarmins; T-Lymphocytes
PubMed: 36439184
DOI: 10.3389/fimmu.2022.941757 -
Marine Drugs Jun 2014A biological screening study of an actinomycetes strain assembly was conducted using a cell-based cytotoxicity assay. The CKK1019 strain was isolated from a sea sand...
A biological screening study of an actinomycetes strain assembly was conducted using a cell-based cytotoxicity assay. The CKK1019 strain was isolated from a sea sand sample. Cytotoxicity-guided fractionation of the CKK1019 strain culture broth, which exhibited cytotoxicity, led to the isolation of chromomycins A2 (1) and A3 (2). 1 and 2 showed potent cytotoxicity against the human gastric adenocarcinoma (AGS) cell line (IC50 1; 1.7 and 2; 22.1 nM), as well as strong inhibitory effects against TCF/β-catenin transcription (IC50 1; 1.8 and 2; 15.9 nM). 2 showed the ability to overcome tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance. To the best of our knowledge, the effects of chromomycins A2 (1) and A3 (2) on TRAIL resistance-overcoming activity, and on the Wnt signaling pathway, have not been reported previously. Thus, 1 and 2 warrant potential drug lead studies in relation to TRAIL-resistant and Wnt signal-related diseases and offer potentially useful chemical probes for investigating TRAIL resistance and the Wnt signaling pathway.
Topics: Actinobacteria; Adenocarcinoma; Antineoplastic Agents; Cell Line, Tumor; Chromomycin A3; Geologic Sediments; Humans; Inhibitory Concentration 50; Plicamycin; Stomach Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Wnt Signaling Pathway
PubMed: 24905484
DOI: 10.3390/md12063466 -
Animal Reproduction Science Mar 2023Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or...
Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or packaging and fertility have not been explored. In the present study, relationships between fertility and DNA fragmentation index, protamine deficiency, total thiols, free thiols and disulfide bonds in stallion spermatozoa were investigated. Ejaculates (n = 36) were collected from 12 stallions and extended to prepare semen doses for insemination. One dose from each ejaculate was sent to the Swedish University of Agricultural Sciences. Aliquots of semen were stained for flow cytometry with acridine orange for the Sperm Chromatin Structure Assay (DNA fragmentation Index, %DFI), with chromomycin A3 (CMA) for protamine deficiency, and with monobromobimane (mBBr) for detection of total and free thiols and disulfide bonds. Per season pregnancy rates after insemination were obtained. Mixed linear models were used to analyze data. Negative correlations were found between pregnancy rate and %DFI (r = -0.35, P < 0.03) and pregnancy rate and free thiols (r = -0.60, P < 0.0001). Furthermore, there were positive correlations between total thiols and disulfide bonds (r = 0.95, P < 0.0001), and protamine and disulfide bonds (r = 0.4100, P < 0.01986). Since chromatin integrity, protamine deficiency and packaging were all associated with fertility, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates.
Topics: Pregnancy; Female; Male; Animals; Horses; Chromatin; Semen; Biofilms; DNA Fragmentation; Bioreactors; Fertility; Spermatozoa; Protamines; Sulfhydryl Compounds; Disulfides
PubMed: 36801727
DOI: 10.1016/j.anireprosci.2023.107200 -
Frontiers in Cell and Developmental... 2023Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and...
Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of and mRNA or in protein levels between testes of wild-type and knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.
PubMed: 37766963
DOI: 10.3389/fcell.2023.1125096 -
Marine Drugs Dec 2014The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and...
The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and demethylchromomycin A2 were also identified from the extract derived from Streptomyces sp., recovered from Paracuru Beach, located in the northeast region of Brazil. The cytotoxic activity of chromomycin A2 was evaluated across a panel of human tumor cell lines, which found IC50 values in the nM-range for exposures of 48 and 72 h. MALME-3M, a metastatic melanoma cell line, showed the highest sensitivity to chromomycin A2 after 48h incubation, and was chosen as a model to investigate this potent cytotoxic effect. Treatment with chromomycin A2 at 30 nM reduced cell proliferation, but had no significant effect upon cell viability. Additionally, chromomycin A2 induced accumulation of cells in G0/G1 phase of the cell cycle, with consequent reduction of S and G2/M and unbalanced expression of cyclins. Chromomycin A2 treated cells depicted several cellular fragments resembling autophagosomes and increased expression of proteins LC3-A and LC3-B. Moreover, exposure to chromomycin A2 also induced the appearance of acidic vacuolar organelles in treated cells. These features combined are suggestive of the induction of autophagy promoted by chromomycin A2, a feature not previously described for chromomycins.
Topics: Autophagy; Brazil; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromomycin A3; Chromomycins; HCT116 Cells; HL-60 Cells; Humans; Melanoma; Microtubule-Associated Proteins; Plicamycin; Streptomyces
PubMed: 25486109
DOI: 10.3390/md12125839 -
Journal of Biological Engineering 2018Regulatory genes play critical roles in natural product biosynthetic pathways. Chromomycins are promising anticancer natural products from actinomycetes. This study is...
BACKGROUND
Regulatory genes play critical roles in natural product biosynthetic pathways. Chromomycins are promising anticancer natural products from actinomycetes. This study is aimed to create an efficient strain for production of these molecules by manipulating the regulatory genes.
RESULTS
A putative but silent chromomycin biosynthetic gene cluster was discovered in . Heterologous expression of the ketosynthase, chain length factor, and acyl carrier protein in confirmed that they are responsible for the assembly of a decaketide. Two regulatory genes are present in this gene cluster, including SARP-type activator SrcmRI and PadR-like repressor SrcmRII. Either overexpression of SrcmRI or disruption of SrcmRII turned on the biosynthetic pathway of chromomycins. The production titers of chromomycin A/A in R5 agar in these two strains reached 8.9 ± 1.2/13.2 ± 1.6 and 49.3 ± 4.3/53.3 ± 3.6 mg/L, respectively. An engineered strain was then constructed with both SrcmRII disruption and SrcmRI overexpression, which produced chromomycins A and A in R5 agar at 69.4 ± 7.6 and 81.7 ± 7.2 mg/L, respectively. Optimization of the culture conditions further increased the titers of chromomycins A and A respectively to 145.1 ± 15.3 and 158.3 ± 15.4 mg/L in liquid fermentation.
CONCLUSIONS
This work revealed the synergistic effect of manipulation of pathway repressor and activator genes in the engineering of a natural product biosynthetic pathway. The resulting engineered strain showed the highest production titers of chromomycins by a strain of , providing an efficient way to produce these pharmaceutically valuable molecules.
PubMed: 29977332
DOI: 10.1186/s13036-018-0103-x -
Frontiers in Endocrinology 2023In this study, the semen parameters, sperm chromatin integrity, antioxidant enzyme levels, and reproductive hormone levels of subfertile male subjects from Pakistan were...
In this study, the semen parameters, sperm chromatin integrity, antioxidant enzyme levels, and reproductive hormone levels of subfertile male subjects from Pakistan were assessed in relation to their age. Data on the demographic characteristics of the 750 study participants, including their general health, body mass index (BMI), and reproductive status, were collected from subfertile men from Pakistan. Semen and blood were collected to determine standard semen parameters, sperm chromatin dispersion (Halosperm-SCD), sperm chromatin integrity using toluidine blue (TB) staining, sperm chromatin maturity using chromomycin A3 (CMA3+) staining, and reproductive hormone (FSH, LH, prolactin and testosterone levels). The patients were divided into three groups according to their age: Group 1 included male subjects aged 30 years or less ( = 90), Group 2 included male subjects between the ages of 31 and 40 years ( = 330), and Group 3 included male subjects over 40 years of age ( = 330). Conventional semen parameters, reactive oxygen species (ROS), superoxide dismutase (SOD), guaiacol peroxidase (GPX), catalase (CAT), and lipid peroxidation (MDA) did not statistically ( > 0.05) differ with increasing male age or between different age groups. When compared to younger men (<30 years), sperm SCD (23.2 ± 0.88%) was significantly ( = 0.01) lower as compared to male patients aged >40 years (26.6 ± 0.6%). The concentration of LH, FSH, and testosterone levels were comparable between the groups ( > 0.05), while a significant ( = 0.04) increase in sperm chromatin immaturity CMA3+ (30 ± 0.71%) was observed in the old age group (>40 years) compared to the <30-year group (26.6 ± 1.03%). A positive association was observed between advanced male age and sperm chromatin dispersion (SCD) ( = 0.124, = 0.001) and decondensation (CMA3+) ( = 0.1, = 0.009). Despite potential limitations, this study has been carried out with extensive information on the potential risk of male age on sperm integrity. The present study demonstrated the impact of male age on male reproductive health, as these patients had a higher percentage of sperm chromatin damage (SCD) in their semen. Sperm DNA damage assessment will help in the evaluation and diagnosis of the underlying cause of poor fertility and can help clinicians in selecting the right treatment options. Male age is one of the factors that have an impact on the decline in male fertility. As a result, it is preferable for patients receiving assisted reproductive technology to be younger.
Topics: Humans; Male; Semen; Chromatin; Infertility, Male; Sperm Count; Sperm Motility; Spermatozoa; Prolactin; Follicle Stimulating Hormone; Testosterone; Biomarkers
PubMed: 37124745
DOI: 10.3389/fendo.2023.1092603 -
Frontiers in Chemistry 2020The TBX2 transcription factor plays critical roles during embryonic development and it is overexpressed in several cancers, where it contributes to key oncogenic...
The TBX2 transcription factor plays critical roles during embryonic development and it is overexpressed in several cancers, where it contributes to key oncogenic processes including the promotion of proliferation and bypass of senescence. Importantly, based on compelling biological evidences, TBX2 has been considered as a potential target for new anticancer therapies. There has therefore been a substantial interest to identify molecules with TBX2-modulatory activity, but no such substance has been found to date. Here, we adopt a targeted approach based on a reverse-affinity procedure to identify the ability of chromomycins A (CA) and A (CA) to interact with TBX2. Briefly, a TBX2-DNA-binding domain recombinant protein was N-terminally linked to a resin, which in turn, was incubated with either CA or CA. After elution, bound material was analyzed by UPLC-MS and CA was recovered from TBX2-loaded resins. To confirm and quantify the affinity (K) between the compounds and TBX2, microscale thermophoresis analysis was performed. CA and CA modified the thermophoretic behavior of TBX2, with a K in micromolar range. To begin to understand whether these compounds exerted their anti-cancer activity through binding TBX2, we next analyzed their cytotoxicity in TBX2 expressing breast carcinoma, melanoma and rhabdomyosarcoma cells. The results show that CA was consistently more potent than CA in all tested cell lines with IC values in the nM range. Of the cancer cell types tested, the melanoma cells were most sensitive. The knockdown of TBX2 in 501mel melanoma cells increased their sensitivity to CA by up to 5 times. Furthermore, inducible expression of TBX2 in 501mel cells genetically engineered to express TBX2 in the presence of doxycycline, were less sensitive to CA than the control cells. Together, the data presented in this study suggest that, in addition to its already recognized DNA-binding properties, CA may be binding the transcription factor TBX2, and it can contribute to its cytotoxic activity.
PubMed: 32195221
DOI: 10.3389/fchem.2020.00110 -
Frontiers in Microbiology 2018Many bioactive natural products are glycosylated compounds in which the sugar components usually participate in interaction and molecular recognition of the cellular...
Many bioactive natural products are glycosylated compounds in which the sugar components usually participate in interaction and molecular recognition of the cellular target. Therefore, the presence of sugar moieties is important, in some cases essential, for bioactivity. Searching for novel glycosylated bioactive compounds is an important aim in the field of the research for natural products from actinomycetes. A great majority of these sugar moieties belong to the 6-deoxyhexoses and share two common biosynthetic steps catalyzed by a NDP--glucose synthase (GS) and a NDP--glucose 4,6-dehydratase (DH). Based on this fact, seventy one strains isolated from the integument of ants of the Tribe were screened for the presence of biosynthetic gene clusters (BGCs) for glycosylated compounds. Total DNAs were analyzed by PCR amplification using oligo primers for GSs and DHs and also for a NDP--glucose-2,3-dehydratases. Amplicons were used in gene disruption experiments to generate non-producing mutants in the corresponding clusters. Eleven mutants were obtained and comparative dereplication analyses between the wild type strains and the corresponding mutants allowed in some cases the identification of the compound coded by the corresponding cluster (lobophorins, vicenistatin, chromomycins and benzanthrins) and that of two novel macrolactams (sipanmycin A and B). Several strains did not show UPLC differential peaks between the wild type strain and mutant profiles. However, after genome sequencing of these strains, the activation of the expression of two clusters was achieved by using nutritional and genetic approaches leading to the identification of compounds of the cervimycins family and two novel members of the warkmycins family. Our work defines a useful strategy for the identification new glycosylated compounds by a combination of genome mining, gene inactivation experiments and the activation of silent biosynthetic clusters in strains.
PubMed: 29441046
DOI: 10.3389/fmicb.2018.00039