Melatonin sensitises shikonin-induced cancer cell death mediated by oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway

Recent research suggests that melatonin (Mel), an endogenous hormone and natural supplement, possesses anti-proliferative effects and can sensitise cells to anti-cancer therapies. Although shikonin (SHK) also possesses potential anti-cancer properties, the poor solubility and severe systemic toxicity of this compound hinders its clinical usage. In this study, we combined Mel and SHK, a potentially promising chemotherapeutic drug combination, with the aim of reducing the toxicity of SHK and enhancing the overall anti-cancer effects. We demonstrate for the first time that Mel potentiates the cytotoxic effects of SHK on cancer cells by inducing oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway. Particularly, Mel-SHK treatment induced oxidative stress, increased mitochondrial calcium accumulation and reduced the mitochondrial membrane potential in various cancer cells, leading to apoptosis. This drug combination also promoted endoplasmic reticulum (ER) stress, leading to AKT dephosphorylation. In HeLa cells, Mel-SHK treatment reduced SIRT3/SOD2 expression and SOD2 activity, while SIRT3 overexpression dramatically reduced Mel-SHK-induced oxidative stress, ER stress, mitochondrial dysfunction and apoptosis. Hence, we propose the combination of Mel and SHK as a novel candidate chemotherapeutic regimen that targets the SIRT3/SOD2-AKT pathway in cancer

Low-calorie sweetener d-psicose promotes hydrogen peroxide-mediated apoptosis in c2c12 myogenic cells favoring skeletal muscle cell injury

Diet and exercise are the most effective approaches used to induce weight loss. D-psicose is a low-calorie sweetener that has been shown to reduce weight in obese individuals. However, the effect of D-psicose on muscle cells under oxidative stress, which is produced during exercise, requires further investigation. The present study aimed to determine the effects of D-psicose on C2C12 myogenic cells in vitro. Hydrogen peroxide (H2O2) was used to stimulate the generation of intracellular reactive oxygen species (ROS) in muscle cells to mimic exercise conditions. Cell viability was analyzed using a MTT assay and flow cytometry was used to analyze the levels of apoptosis, mitochondrial membrane potential (MMP), the generation of ROS and the cell cycle distribution following treatment. Furthermore, protein expression levels were analyzed using western blotting and cell proliferation was determined using a colony formation assay. The results of the present study revealed that D-psicose alone exerted no toxicity on C2C12 mouse myogenic cells. However, in the presence of low-dose (100 μM) H2O2-induced ROS, D-psicose induced C2C12 cell injury and significantly decreased C2C12 cell viability in a dose-dependent manner. In addition, the levels of apoptosis and the generation of ROS increased, while the MMP decreased. MAPK family molecules were also activated in a dose-dependent manner following treatment. Notably, the combined treatment induced G2/M phase arrest and reduced the proliferation of C2C12 cells. In conclusion, the findings of the present study suggested that D-psicose may induce toxic effects on muscle cells in a simulated exercise situation by increasing ROS levels, activating the MAPK signaling pathway and disrupting the MMP.</p

Low-calorie sweetener d-psicose promotes hydrogen peroxide-mediated apoptosis in c2c12 myogenic cells favoring skeletal muscle cell injury

Diet and exercise are the most effective approaches used to induce weight loss. D-psicose is a low-calorie sweetener that has been shown to reduce weight in obese individuals. However, the effect of D-psicose on muscle cells under oxidative stress, which is produced during exercise, requires further investigation. The present study aimed to determine the effects of D-psicose on C2C12 myogenic cells in vitro. Hydrogen peroxide (H2O2) was used to stimulate the generation of intracellular reactive oxygen species (ROS) in muscle cells to mimic exercise conditions. Cell viability was analyzed using a MTT assay and flow cytometry was used to analyze the levels of apoptosis, mitochondrial membrane potential (MMP), the generation of ROS and the cell cycle distribution following treatment. Furthermore, protein expression levels were analyzed using western blotting and cell proliferation was determined using a colony formation assay. The results of the present study revealed that D-psicose alone exerted no toxicity on C2C12 mouse myogenic cells. However, in the presence of low-dose (100 μM) H2O2-induced ROS, D-psicose induced C2C12 cell injury and significantly decreased C2C12 cell viability in a dose-dependent manner. In addition, the levels of apoptosis and the generation of ROS increased, while the MMP decreased. MAPK family molecules were also activated in a dose-dependent manner following treatment. Notably, the combined treatment induced G2/M phase arrest and reduced the proliferation of C2C12 cells. In conclusion, the findings of the present study suggested that D-psicose may induce toxic effects on muscle cells in a simulated exercise situation by increasing ROS levels, activating the MAPK signaling pathway and disrupting the MMP.</p

Seasonal Variation and Global Public Interest in the Internet Searches for Osteoporosis

Background. To ascertain the seasonal pattern and global public interest in osteoporosis by evaluating search term popularity changes of the disease over a decade. Methods. We applied Google Trends to retrieve search popularity scores for the term “osteoporosis” between January 01, 2004, and December 31, 2019. Cosinor analyses were conducted to examine the seasonality of osteoporosis, and analysis on osteoporosis-related topics including hot topics and rising-related topics was also performed. Results. The cosinor analyses demonstrated a statistically significant seasonal variation in relative search volume of the “osteoporosis” in the world (p=0.0083), USA (p<0.001), UK (p<0.001), Canada (p<0.001), Ireland (p<0.001), Australia (p<0.001), and New Zealand (p<0.001), with a peak in the late winter months and trough in the summer months. The peaks in late winter and valley in summer presented an approximately 6-month difference between hemispheres. The top 11 rising topics were denosumab, FRAX, hypocalcaemia, zoledronic acid, ibandronic acid, osteomyelitis, osteopenia, osteoarthritis, bone, calcium, and bone density. Conclusions. Google search query volumes related to osteoporosis follow strong seasonal patterns with late winter peaks and summer troughs. Further studies aimed at elucidating the possible mechanisms behind seasonality in osteoporosis are needed. Moreover, Internet data including the top rising topics may alert physicians to strengthen the propaganda of osteoporosis timely, so as to further promote the development of public health interventions

Analysis of microRNA expression in CD133 positive cancer stem‑like cells of human osteosarcoma cell line MG-63

Osteosarcoma (OS) is a primary malignant tumor of bone occurring in young adults. OS stem cells (OSCs) play an important role in the occurrence, growth, metastasis, drug resistance and recurrence of OS. CD133 is an integral membrane glycoprotein, which has been identified as an OSC marker. However, the mechanisms of metastasis, chemoresistance, and progression in CD133(+) OSCs need to be further explored. In this study, we aim to explore differences in miRNA levels between CD133(+) and CD133(−) cells from the MG-63 cell line. We found 20 differentially expressed miRNAs (DEmiRNAs) (16 upregulated and 4 downregulated) in CD133(+) cells compared with CD133(−) cells. Hsa-miR-4485-3p, hsa-miR-4284 and hsa-miR-3656 were the top three upregulated DEmiRNAs, while hsa-miR-487b-3p, hsa-miR-493-5p and hsa-miR-431-5p were the top three downregulated DEmiRNAs. In addition, RT-PCR analysis confirmed that the expression levels of hsa-miR-4284, hsa-miR-4485-3p and hsa-miR-3656 were significantly increased, while the expression levels of hsa-miR-487b-3p, hsa-miR-493-5p, and hsa-miR-431-5p were significantly decreased in CD133(+) cells compared with CD133(−) cells. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that predicted or validated target genes for all 20 DEmiRNAs or the selected 6 DEmiRNAs participated in the “PI3K-Akt signaling pathway,” “Wnt signaling pathway,” “Rap1 signaling pathway,” “Cell cycle” and “MAPK signaling pathway”. Among the selected six DEmiRNAs, miR-4284 was especially interesting. MiR-4284 knockdown significantly reduced the sphere forming capacity of CD133(+) OS cells. The number of invasive CD133(+) OS cells was markedly decreased after miR-4284 knockdown. In addition, miR-4284 knockdown increased the p-β-catenin levels in CD133(+) OS cells. In conclusion, RNA-seq analysis revealed DEmiRNAs between CD133(+) and CD133(−) cells. MiRNAs might play significant roles in the function of OSCs and could serve as targets for OS treatment. MiR-4284 prompted the self-renewal and invasion of OSCs. The function of miR-4284 might be associated with the Wnt signaling pathway

Melatonin sensitises shikonin-induced cancer cell death mediated by oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway

Recent research suggests that melatonin (Mel), an endogenous hormone and natural supplement, possesses anti-proliferative effects and can sensitise cells to anti-cancer therapies. Although shikonin (SHK) also possesses potential anti-cancer properties, the poor solubility and severe systemic toxicity of this compound hinders its clinical usage. In this study, we combined Mel and SHK, a potentially promising chemotherapeutic drug combination, with the aim of reducing the toxicity of SHK and enhancing the overall anti-cancer effects. We demonstrate for the first time that Mel potentiates the cytotoxic effects of SHK on cancer cells by inducing oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway. Particularly, Mel-SHK treatment induced oxidative stress, increased mitochondrial calcium accumulation and reduced the mitochondrial membrane potential in various cancer cells, leading to apoptosis. This drug combination also promoted endoplasmic reticulum (ER) stress, leading to AKT dephosphorylation. In HeLa cells, Mel-SHK treatment reduced SIRT3/SOD2 expression and SOD2 activity, while SIRT3 overexpression dramatically reduced Mel-SHK-induced oxidative stress, ER stress, mitochondrial dysfunction and apoptosis. Hence, we propose the combination of Mel and SHK as a novel candidate chemotherapeutic regimen that targets the SIRT3/SOD2-AKT pathway in cancer.</p

Integration analysis of lncRNA and mRNA expression data identifies DOCK4 as a potential biomarker for elderly osteoporosis

Abstract Background We aimed to identify some potential biomarkers for elderly osteoporosis (OP) by integral analysis of lncRNA and mRNA expression data. Methods A total of 8 OP cases and 5 healthy participants were included in the study. Fasting peripheral venous blood samples were collected from individuals, and total RNA was extracted. RNA-seq library was prepared and sequenced on the Illumina HiSeq platform. Differential gene expression analysis was performed using “DESeq2” package in R language. Functional enrichment analysis was conducted using the “clusterProfiler” package, and the cis- and trans-regulatory relationships between lncRNA and target mRNA were analyzed by the lncTar software. A protein-protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified through the MCODE plugin in Cytoscape. Results We identified 897 differentially expressed lncRNAs (DELs) and 1366 differentially expressed genes (DEGs) between normal and OP samples. After co-expression network analysis and cis-trans regulatory genes analysis, we identified 69 candidate genes regulated by lncRNAs. Then we further screened 7 genes after PPI analysis. The target gene DOCK4, trans-regulated by two lncRNAs, was found to be significantly upregulated in OP samples. Additionally, 4 miRNAs were identified as potential regulators of DOCK4. The potential diagnostic value of DOCK4 and its two trans-regulatory lncRNAs was supported by ROC analysis, indicating their potential as biomarkers for OP. Conclusion DOCK4 is a potential biomarker for elderly osteoporosis diagnostic. It is identified to be regulated by two lncRNAs and four miRNAs

Protective effects of baicalin against phenylarsine oxide-induced cytotoxicity in human skin keratinocytes

Phenylarsine oxide (PAO) is a known environmental pollutant and skin keratinocytes are most seriously affected. Baicalin (BCN) was reported to have antioxidant and anti-inflammatory effects, but its protective effect against PAO toxicity is unknown. This study aimed at exploring whether baicalin can reverse the toxicity of human epidermal keratinocytes that are subjected to PAO exposure and underlying mechanisms. In silico analysis from a publicly accessible HaCaT cell transcriptome dataset exposed to chronic Arsenic showed significant differential expression of several genes, including the genes related to DNA replication. Later, we performed in vitro experiments, in which HaCaT cells were exposed to PAO (500 nM) in the existence of BCN (10–50 µM). Treatment of PAO alone induces the JNK, p38 and caspase-3 activation, which were engaged in the apoptosis induction, while the activity of AKT was significantly inhibited, which was engaged in the suppression of apoptosis. PAO suppressed SIRT3 expression and induced intracellular reactive oxygen species (ROS), causing a marked reduce in cell viability and apoptosis. However, BCN treatment restored the PAO-induced suppression of SIRT3 and AKT expression, reduced intracellular ROS generation, and markedly suppressed both caspase-3 activation and apoptosis induction. However, the protective effect of BCN was significantly attenuated after pretreatment with nicotinamide, an inhibitor of SIRT3. These findings indicate that BCN protects against cell death induced by PAO via inhibiting excessive intracellular ROS generation via restoring SIRT3 activity and reactivating downstream AKT pathway. In this study, we firstly shown that BCN is an efficient drug to prevent PAO-induced skin cytotoxicity, and these findings need to be confirmed by in vivo and clinical investigations.</p

Inhibition of HIF-1α by siRNA results in downregulation of Sost expression in osteoblasts.

<p>MC3T3 osteoblasts were transfected with siRNA control or siRNA against HIF-1α. RNA was isolated 24 hr post-transfection and quantitated by quantitative real-time RT-PCR for HIF-1α and Sost, and HSP90 was used as a negative control. The RNA level from the control siRNA group was normalized to a value of 1. Values were presented as the mean ±S.D. si-control: si-RNA control; si-HIF-1α: si-RNA against HIF-1α. A paired <i>t</i>-test was performed comparing si-control group and si-HIF-1α group. *: A star indicates statistical significance compared to control group.</p

Effect of HIF-1α on Sost promoter activity.

<p>(A) HIF-1α activates the <i>Sost</i> promoter in a dose-dependent manner. HEK293 cells were transfected with a 1 kb <i>Sost</i> promoter-luciferase reporter gene without or with increasing amounts of an HIF-1α-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D. (B) Jab1 does not activate <i>Sost</i> promoter activity. HEK293 cells were transfected with a 1 kb <i>Sost</i> promoter-luciferase reporter gene without or with increasing amounts of a Jab1-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D.</p