Decreased Cell Proliferation Rate Research Articles

Allogeneic bone marrow mesenchymal stem cell-derived exosomes alleviate human hypoxic AKI-on-a-Chip within a tight treatment window

BackgroundAcute hypoxic proximal tubule (PT) injury and subsequent maladaptive repair present high mortality and increased risk of acute kidney injury (AKI) - chronic kidney disease (CKD) transition. Human bone marrow mesenchymal stem cell-derived exosomes (hBMMSC-Exos) as potential cell therapeutics can be translated into clinics if drawbacks on safety and efficacy are clarified. Here, we determined the real-time effective dose and treatment window of allogeneic hBMMSC-Exos, evaluated their performance on the structural and functional integrity of 3D microfluidic acute hypoxic PT injury platform.MethodshBMMSC-Exos were isolated and characterized. Real-time impedance-based cell proliferation analysis (RTCA) determined the effective dose and treatment window for acute hypoxic PT injury. A 2-lane 3D gravity-driven microfluidic platform was set to mimic PT in vitro. ZO-1, acetylated α-tubulin immunolabelling, and permeability index assessed structural; cell proliferation by WST-1 measured functional integrity of PT.ResultshBMMSC-Exos induced PT proliferation with ED50 of 172,582 µg/ml at the 26th hour. Hypoxia significantly decreased ZO-1, increased permeability index, and decreased cell proliferation rate on 24–48 h in the microfluidic platform. hBMMSC-Exos reinforced polarity by a 1.72-fold increase in ZO-1, restored permeability by 20/45-fold against 20/155 kDa dextran and increased epithelial proliferation 3-fold compared to control.ConclusionsThe real-time potency assay and 3D gravity-driven microfluidic acute hypoxic PT injury platform precisely demonstrated the therapeutic performance window of allogeneic hBMMSC-Exos on ischemic AKI based on structural and functional cellular data. The novel standardized, non-invasive two-step system validates the cell-based personalized theragnostic tool in a real-time physiological microenvironment prior to safe and efficient clinical usage in nephrology.

Targeting Breast Cancer with N-Acetyl-D-Glucosamine: Integrating Machine Learning and Cellular Assays for Promising Results.

Breast cancer is a common cancer with high mortality rates. Early diagnosis is crucial for reducing the prognosis and mortality rates. Therefore, the development of alternative treatment options is necessary. This study aimed to investigate the inhibitory effect of N-acetyl-D-glucosamine (D-GlcNAc) on breast cancer using a machine learning method. The findings were further confirmed through assays on breast cancer cell lines. MCF-7 and 4T1 cell lines (ATCC) were cultured in the presence and absence of varying concentrations of D-GlcNAc (0.5 mM, 1 mM, 2 mM, and 4 mM) for 72 hours. A xenograft mouse model for breast cancer was established by injecting 4T1 cells into mammary glands. D-GlcNAc (2 mM) was administered intraperitoneally to mice daily for 28 days, and histopathological effects were evaluated at pre-tumoral and post-tumoral stages. Treatment with 2 mM and 4 mM D-GlcNAc significantly decreased cell proliferation rates in MCF-7 and 4T1 cell lines and increased Fas expression. The number of apoptotic cells was significantly higher than untreated cell cultures (p < 0.01 - p < 0.0001). D-GlcNAc administration also considerably reduced tumour size, mitosis, and angiogenesis in the post-treatment group compared to the control breast cancer group (p < 0.01 - p < 0.0001). Additionally, molecular docking/dynamic analysis revealed a high binding affinity of D-GlcNAc to the marker protein HER2, which is involved in tumour progression and cell signalling. Our study demonstrated the positive effect of D-GlcNAc administration on breast cancer cells, leading to increased apoptosis and Fas expression in the malignant phenotype. The binding affinity of D-GlcNAc to HER2 suggests a potential mechanism of action. These findings contribute to understanding D-GlcNAc as a potential anti-tumour agent for breast cancer treatment.

Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation.

Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH. Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH. Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling. A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.

Effects of Tricalcium Silicate Cement on Corneal Cell Proliferation and Its Relationship with Vascular Endothelial Growth Factor Level and Receptor Typing

This research analyzed the effects of tricalcium silicate (C3S) cement and hypoxia on proliferation of human corneal epithelial cells (HCEpiCs) and the levels of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). Nanoscale C3S was prepared using a combustion method and characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), and laser particle size (LPS) analyzer. HCEpiCs were cultured, and influences of C3S with changed concentrations on proliferation of (HCEpiCs were analyzed. The cells were treated with hypoxia or with low-concentration (0.5 mg/mL, LC-C3S), medium-concentration (5 mg/mL, MC-C3S), or high-concentration (50 mg/mL, HC-C3S) of C3S. Meanwhile, normal HCEpiCs were undertaken as controls (Ctrl group). Cell proliferation, apoptosis, and the expression of target genes were detected using CCK-8, Annexin V-FITC/PI, fluorescent quantitative polymerase chain reaction (fqPCR), and Western blotting (WB). The results suggested that nanoscale C3S had multiple morphologies and an average particle size (APS) of (231.5±8.3) nm. With increasing nanoscale C3S concentration, proliferation of HCEpiCs increased (P &lt; 0.05), and the highest proliferation was visualized at 5 mg/mL. Based on the conditions in the Ctrl group, the hypoxia group exhibited a decreased proliferation rate (PR), an increased apoptosis rate (AR), downshifted VEGF, VEGFR-2, and VEGFR-3, and elevated VEGFR-1 (P &lt; 0.05). Based on the hypoxia group, the LCC3S, MC-C3S, and HC-C3S groups presented increased cell PRs, decreased APs, upshifted VEGF, VEGFR-2, and VEGFR-3, and downregulated VEGFR-1 (P &lt; 0.05). The MC-C3S group showed an increased cell PR, a decreased AP, upregulated VEGF, VEGFR-2, and VEGFR-3, and downregulated VEGFR-1 to the LC-C3S group (P &lt; 0.05). Additionally, the HC-C3S group had a decreased cell PR, an increased AP, upregulated VEGF, VEGFR-2, and VEGFR-3, and a downshifted VEGFR-1 to the MC-C3S group (P &lt; 0.05). Therefore, C3S promoted proliferation of HCEpiCs, upregulated VEGF, VEGFR-2, and VEGFR-3, and downregulated VEGFR-1.

Upregulation of lncRNA PTOV1-AS1 in hepatocellular carcinoma contributes to disease progression and sorafenib resistance through regulating miR-505.

Increasing evidence has displayed the vital influence of lncRNA in tumorigenesis and chemoresistance of cancer treatment. This study investigated the function of lncRNA PTOV1-AS1 in hepatocellular carcinoma (HCC) and its role in sorafenib resistance.The relative expression of lncRNA and miRNA was measured by RT-qPCR. The cellular activities including cell proliferation and invasion were explored by CCK-8 and Transwell assays. Bioinformatics analysis and dual-luciferase reporter assay were used to predict the targeting miRNA of PTOV1-AS1.The expression levels of PTOV1-AS1 were higher in HCC tissues than that in the normal tissues and associated with patients'overall survival. Knockdown of PTOV1-AS1 decreased cell proliferation rate and invasion number. After treatment with different concentrations of sorafenib, the sorafenib-resistant hepatoma cells were conducted. PTOV1-AS1 expression levels were increased in HepG2-SR and Huh7-SR cells. PTOV1-AS1 knockdown repressed the proliferation, invasion, and drug resistance of sorafenib-resistant HCC cells by targeting the expression of miR-505.In conclusion, the expression of PTOV1-AS1 is increased in HCC and sorafenib-resistance HCC cells, as well as is associated with patients'prognosis. Inhibition of PTOV1-AS1 expression can reduce the resistance of sorafenib-resistant HCC cells, which may play a role by targeting the negative regulation of miR-505 expression.

Arum palaestinum delays hepatocellular carcinoma proliferation through the PI3K-AKT-mTOR signaling pathway and exhibits anticoagulant effects with antimicrobial properties.

Background: Arum palaestinum Boiss (AP) is a wild plant in Palestine whose leaves have a long history as food and medicine in Middle Eastern countries. The current study aimed to evaluate the biological characteristics of AP flower extract, including its antimicrobial and coagulation cascade activities and its effects on anticancer molecular pathways. Methods: The antimicrobial activity of the aqueous extract of AP flowers was assessed using a microdilution assay against eight pathogens. The coagulation properties were assessed by prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT) tests using standard hematological methods. The biological effects of AP on hepatocellular carcinoma were measured by assessing the impact of AP on cell cycle, proliferation (CFSE), apoptosis (annexin-v+/PI), and tumorigenicity (αFP and HBsAg), as well as its effects on the PI3K-AKT-mTOR molecular signaling pathway. Results: The antimicrobial screening results revealed that the aqueous extract of AP had potent antibacterial effects against P. vulgaris and E. faecium compared to ampicillin, with MIC values of 6.25, 6.25, and 18μg/mL, respectively. Moreover, the AP aqueous extract exerted anticoagulant activity, with significant prolonged results in the aPTT and TT tests (25μg/mL and 50μg/mL, respectively) and slightly prolonged results in the PT test (50μg/mL). The anticancer results indicated a delay in the cell cycle through decreased cell proliferation rates following incubation with AP fractions. The effect of the aqueous fraction was most evident in a delay in the S phase. The aqueous and DMSO fractions maintained the cells in the G2-M phase, similar to the DOX, while the flower extract in methanol accelerated the cells in the G2-M phase, suggesting that AF flower extracts may have anti-cancer properties. The aqueous extract of AP 1) reduced secretions of HCC αFP by 1.55-fold and 3.3-fold at the 50 and 100μg/mL concentrations, respectively (p = 0.0008); 2) decreased phosphorylation in the PI3K-AKT-mTOR signaling pathway (p < 0.05); and 3) shifted cells from necrosis to apoptosis by 50% and 70% at the 50 and 100μg/mL concentrations, respectively (p < 0.05). Conclusion: The results of this study showed the activities of the bioactive components for the treatment of infectious diseases and blood coagulation disorders, which could also be a potential therapeutic approach for delaying HCC tumorigenicity.

Ironomycin Induces Mantle Cell Lymphoma Cell Death By Targeting Iron Metabolism Addiction

Mantle-cell lymphoma (MCL) accounts for approximately 5 to 7% of all lymphomas, and in North America and Europe. MCL remains an aggressive and incurable cancer with existing therapies, presenting a significant unmet clinical need. Accumulating evidence reveals that abnormal iron metabolism plays an important role in tumorigenesis and in cancer progression of many tumors. In cancer cells, the demand for iron increases in response to sustained deregulation of cell proliferation and DNA synthesis. Based on these data, we searched to identify alterations of iron homeostasis in MCL that could be exploited to develop novel therapeutic strategies. Analysis of the iron metabolism gene expression profile of a cohort of patients with MCL (n=71) established the iron score (IS), a gene expression-based risk score enabling identification of patients with MCL with a poor outcome who might benefit from a suitable targeted therapy. We analyzed the therapeutic interest of ironomycin, a new promising iron depleting molecule. Ironomycin is known to sequester iron in the lysosome and to induce ferroptosis (Mai TT et al. Nature Chemistry 2017). Preclinical studies in Acute Myeloid Leukemia (Garciaz et al., 2022 Cancer Discovery) and Diffuse Large B-Cell Lymphoma (Devin et al., 2022 Can Res) have revealed the therapeutic interest of ironomycin in treating hematological malignancies. In a panel of 6 MCL cell lines, ironomycin inhibited MCL cell growth at nanomolar concentrations compared with typical iron chelators. Then, we investigated if ironomycin induces cell death through apoptosis. Flow cytometry analysis revealed that ironomycin treatment resulted in AnnV+/7AAD+ and AnnV+/7AAD- death profiles in MCL. This effect was partially inhibited by the pan-caspase inhibitor Q-VD-Oph. Ironomycin also induced caspases 8 and 9 cleavage, and the activation of caspases 3/7. Furthermore, we found that ironomycin induces ferroptosis with lipid peroxidation and the production of ROS monitored by the fluorescence probe CM-H2DCFDA. Ironomycin induced cell death is inhibited by the ferroptosis inhibitor, ferrostatin-1. The treatment did not induce ferritinophagy, as there was no significant change on the expression of LC3B-II, ferritin and the transferrin receptor (CD71).Taken together, these data indicate that ironomycin kills MCL cells by apoptosis mediated by caspase activation and ferroptosis. Ironomycin significantly decreased cell proliferation rate, with a reduced percentage of cells in S-phase together with Ki67 and Cyclin D1 downregulation. Western blot analysis showed an increase of Chk1 and Chk2 phosphorylation, two central factors of DNA damage response, and an increase of the phosphorylation of γ-H2AX, indicative of DNA damage. With major importance, we validated the therapeutic interest of ironomycin in primary MCL cells of patients (n=5). Ironomycin (20 nM) demonstrated a significant higher toxicity in MCL cells compared to normal cells from the microenvironment. Furthermore, ironomycin presented a low toxicity on hematopoietic progenitors (CFU assays, n=5) compared to conventional treatment. We tested the therapeutic interest to combine ironomycin with conventional chemotherapy used in MCL. Interestingly, we identified a synergistic effect when ironomycin is combined with Ibrutinib BTK inhibitor. Furthermore, Significant synergistic effects were also observed by combining ironomycin with BH3 mimetics or Doxorubicin. Altogether, these data underline that MCL patients my benefit from targeting iron homeostasis using ironomycin alone or in combination with conventional MCL treatments.

The effect of aging on the repeated-dose liver micronucleus assay using diethylnitrosamine

BackgroundThe repeated-dose liver micronucleus (RDLMN) assay has been well-developed and applied because of its simplicity and the ease of integration into general toxicity studies which is the preferred method from the 3R’s point of view. In this assay, we observed micronucleated hepatocytes which accumulated during a rather long-term dosing period. When considering integration into general toxicity studies, the effects of age of the animals used in the micronucleus assay becomes a major issue. The effect of age on the micronucleus induction rate has been reported in bone marrow micronucleus assays, and it is considered that the decrease in cell proliferation rate due to aging is the cause of the decrease in sensitivity. A decrease in sensitivity due to aging was also reported in a liver micronucleus assay using clofibrate and the cause is considered to be a decrease in hepatocyte proliferation activity due to aging. However, no actual decrease in hepatocyte proliferation rate due to aging has been reported. In addition, there are no reports, so far, on whether similar effects of aging appear when other substances were administered. To investigate the effects of aging in the RDLMN assay, this study focused on the effects of 14-day repeated administration of DEN, a well-known genotoxic hepatocarcinogen with the hepatocyte toxicity which should cause an elevation of cell proliferation rate as a reflective regeneration.ResultsThe liver micronuclei induced by DEN were equivalent between the two age groups (i.e., six and eight weeks of age at the start of dosing). In the histopathological examination for the liver, single cell necrosis, karyomegaly, and increased mitosis were observed in the hepatocytes, and the frequency and severity were increased dose-dependently. Ki-67 immunohistochemical analysis which can detect all cells in the cell cycle other than those in the G0 phase revealed dose-dependent increase of cell proliferation activity, and the difference between ages was not observed.ConclusionThe effect of aging on the RDLMN assay could not be recognized when DEN was administered for 14 days in rats. Meanwhile, it was supported by the histopathological examination and Ki-67 immunohistochemical analysis that such an effect of aging was masked by the compensatory hepatocyte proliferation which was induced by the hepatocyte toxicity of DEN.

Inhibition of TRPM7 suppresses migration and invasion of prostate cancer cells via inactivation of ERK1/2, Src and Akt pathway signaling

Background: Prostate cancer is the second most common cause of cancer related death in males worldwide. Most patients show no response to androgen deprivation therapy in case of recurrence and proceed to advanced stage with metastasis. TRPM7 is reported to be upregulated in diverse types of tumors. Methods: We analyzed the expression of TRPM7 and related proteins by Western blotting analysis. We performed cell migration and invasion assay to analyze the relationship between tumor aggressiveness and TRPM7. In addition, we proceeded an animal study by using stable TRPM7 knockdown cell line in xenograft. Results: In our results, TRPM7 regulates prostate cancer cell biology including proliferation, migration and invasion through ERK1/2, PI3K/Akt and JNK signaling pathways. We produced stable TRPM7 knockdown prostate cancer cell line. To analyze the relationship between TRPM7 and tumorigenesis, we proceeded migration and invasion assay as well as xenograft model. TRPM7 down-regulated DU145 cells showed suppressed migratory and invasion ability, 0.65- and 0.05-fold, respectively. In addition, we confirmed that the anti-cancer effect of TRPM7 is mediated through inactivation of ERK1/2, Src and Akt signaling pathways by western blotting analysis. P-ERK1/2, p-Src, and p-Akt expressions were reduced to 0.66-, 0.68-, and 0.66-fold, respectively. Moreover, we treated ERK, Akt and Src inhibitors to clarify the involvement of related each protein in migration and invasion ability, and we could observe that inhibitor treated cells showed suppressed migration and invasion ability. In vivo, TRPM7 knockdown cells projected decreased cell proliferation rate. Conclusions: Taken these results together, out study suggested TRPM7 might be an essential gene for prostate cancer metastasis by regulating prostate cancer cell proliferation, migration and invasion ability.

MiR-4323 targets hepatoma-derived growth factor (HDGF) to suppress colorectal cancer cell proliferation

MicroRNAs (miRNAs) are regulators of cancer progression via directly binding to the 3′ untranslated region (3′UTR) of target genes to control the activity of signaling network. Recent studies have revealed the function of several miRNAs in colorectal cancer, however, there are still numerous miRNAs which have not been studied yet. Herein, we showed that miR-4323 was a downregulated miRNA according to previous microarray data. The downregulation of miR-4323 was further confirmed in colorectal tumors via RT-qPCR. miR-4323 overexpression decreased cell proliferation rate via induction of cell apoptosis in colorectal cancer cells. Mechanistically, miR-4323 decreased β-catenin and its downstream genes including c-Myc and MMP9 in colorectal cancer cells, indicating the inactivation of Wnt signaling. HDGF, an anti-apoptotic protein, was predicted by several software as a potential target of miR-4323. HDGF was experimentally verified as a target gene of miR-4323 using dual luciferase reporter assay. Ectopic expression of HDGF attenuated the effect of miR-4323 on cell proliferation and apoptosis in cells. Altogether, the data demonstrate a critical role of miR-4323 in the regulation of colorectal cancer.

SOCS5 Regulates JAK-STAT Signaling and T-ALL Development

T-lineage acute lymphoblastic leukemia (T-ALL) is a hematopoietic disease that tends to present with high-risk features in children, adolescents and young adults. While cure rates have improved in recent decades, event free survival is <10% in patients with relapsed disease. Activating mutations in IL-7, JAK1, JAK2, JAK3 have been implicated in aberrant JAK/STAT signal transduction and disease progression. However, the roles played by suppressors of the cytokine signaling (SOCS) remain incompletely understood. Because SOCS5 plays a key regulatory role in T-helper (Th) cell differentiation, we hypothesized that this gene is a critical determinant in mediating aberrant JAK/STAT signaling in T-ALL. To test regulatory effects of SOCS5 on T-ALL signal transduction and progression, we utilized lentiviral shRNA-mediated methods to knock down of SOCS5 in PF382 and KoptK1 T-ALL cell lines or, alternatively, to force over-expression of SOCS5 in ALL-SIL cells, respectively. We found that SOCS5 silencing resulted in enhanced cellular proliferation (P < 0.05), and that forced expression of SOCS5 resulted in significant decrease in cell proliferation rate (p < 0.05), suggesting that SOCS5 negatively regulates T-ALL cell growth in vitro. We utilized flow-based assays to examine the effects of SOCS5 on cell cycle control, and found that SOCS5 depletion significantly increased cell cycle progression in S/G2/M phases and decreased G1 (p < 0.05), but had no effect on apoptotic cell death. Forced expression of SOCS5 delayed cell cycle progression as demonstrated by increased G1 and decreased S/G2/M phases of cell cycle (p < 0.05). We next utilized protein analyses to examine the effects of SOCS5 on JAK-STAT signaling and found that suppressed SOCS5 expression induced phosphorylation of STAT1, STAT5, STAT6 while forced SOCS5 expression decreased STATs activation. Because STAT6 is involved in regulation of IL4 signaling we tested whether SOCS5 regulates the expression of this cytokine and its receptor, IL4R and demonstrated that SOCS5 depletion upregulated IL4 and its receptor in CCRF-CEM and KoptK1 cells. We next asked whether SOCS5 regulates IL7 signaling, which is critical for T-ALL development and resistance. Down-regulation of SOCS5 expression enhanced expression of both, IL7 and IL7R. On the contrary, SOCS5 overexpression decreased the levels of IL7 and IL7R. These findings suggest that SOCS5 is involved in negative regulation of IL4 and IL7 signaling. Similar experiments showed the SOCS5 leads to aberrant signaling and expression of c-myc oncogene. To study the effect of SOCS5 on leukemia development in a murine xenograft model, PF382 cells transduced with SOCS5 shRNA and control plasmid were transplanted intravenously into the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (10 mice per group) followed by survival analyses. We observed a significant decrease in mouse survival after SOCS5 depletion (median survival - 21 days) compared to animals engrafted with negative control cells (median survival - 30 days; p < 0.0002), indicating that SOCS5 inactivation promotes T-ALL progression. Using the same murine model, depletion of SOCS5 significantly increased the number of leukemic cells in bone marrow (92.4 ± 3.5 % vs. 13.6 ± 6 %; p < 0.0001), brain (88.4 ± 1.5 % vs. 14.7 ± 5.6 %; p < 0.0001) and liver (49.5 ± 8.2 % vs. 6.1 ± 1.5 %; p < 0.002) compared to negative control group. Our results link the aberrant SOCS5 expression to the up-regulation of the JAK-STAT and cytokine receptor-signaling cascade in T-ALL. We propose deregulation of SOCS5 expression as a novel mechanism governing aberrant cytokine signaling and JAK-STAT signal transduction in T-ALL. We conclude that SOCS5 is an important regulator of T-ALL cell proliferation and leukemic progression in sanctuary and non-sanctuary sites. DisclosuresNo relevant conflicts of interest to declare.

Anticancer, Free Radicals, and Digestive Enzyme Inhibitory Activities of Rubus sanctus Schreb Root Four Solvent Fractions.

Background Humankind used herbal products as a source of medicines since they understood their therapeutic benefits from ancient times. Therefore, the current research aimed to determine the anticancer, antioxidant, and metabolic enzyme inhibitory activities of Rubus sanctus (RS) root four solvent fractions for the first time. Methods The antioxidant, antilipase, and anti-α-amylase potentials of (RS) four solvent fractions were evaluated using standard biomedical assays. Moreover, the DNA cell cycle of liver cancer was assessed using a propidium iodide (PI) assay. At the same time, the apoptosis activity was estimated utilizing flow the cytometry method. Results The methanol and acetone (RS) fractions showed the highest antioxidant activity with IC50 values of 0.078 ± 0.22 and 0.67 ± 0.25 μg/ml, respectively, compared with Trolox, which has an antioxidant IC50 value of 2.039 ± 0.52 μg/ml. Moreover, the methanol (RS) fraction has the highest anti-α-amylase activity with an IC50 value of 20.12 ± 0.34 μg/ml compared with acarbose, which has an IC50 value of 6.565 ± 0.3 μg/ml. Also, the acetone (RS) fraction revealed the highest antilipase activity with an IC50 value of 6.03 ± 1.23 μg/ml compared with the positive control orlistat which has an IC50 value of 0.39 ± 0.45 μg/ml. The aqueous, methanol, acetone, and hexane fractions of the (RS) roots decreased the secretion of the α-fetoprotein in the liver cancer cells. The acetone fraction was the most potent α-fetoprotein inhibitor with an average of 237 ± 12.5% compared with the average of the untreated cells, which was 4066.6 ± 202%. The hexane fraction was the most effective in diminishing apoptosis with an average of 14.5 ± 1.6%, compared with 49% ± 2 untreated cells' average. In inhibiting cell cycle progression, it was recognized that methanol fraction seems to be the most powerful amplifier of the (RS) effect, as it increased the proportion of the cells with an average of 24.5 ± 2.2%, compared with 7.4 ± 1.8% in the doxorubicin (DOX). Data indicated a decrease in cell proliferation rate by prolonging the G2-M phase and thus slowing cancer progression. Our results suggest that (RS) roots four solvent fractions have potential anticancer activity. Conclusion The (RS) roots four solvent fractions have potential anticancer, antioxidant, antilipase, and α-amylase inhibitory activities. It could be a promising source for applications in the functional food, nutraceutical, and pharmaceutical industries.

α-Chaconine Affects the Apoptosis, Mechanical Barrier Function, and Antioxidant Ability of Mouse Small Intestinal Epithelial Cells.

α-Chaconine is the most abundant glycoalkaloid in potato and toxic to the animal digestive system, but the mechanisms underlying the toxicity are unclear. In this study, mouse small intestinal epithelial cells were incubated with α-chaconine at 0, 0.4, and 0.8 μg/mL for 24, 48, and 72 h to examine apoptosis, mechanical barrier function, and antioxidant ability of the cells using a cell metabolic activity assay, flow cytometry, Western blot, immunofluorescence, and fluorescence quantitative PCR. The results showed that α-chaconine significantly decreased cell proliferation rate, increased apoptosis rate, decreased transepithelial electrical resistance (TEER) value, and increased alkaline phosphatase (AKP) and lactate dehydrogenase (LDH) activities, and there were interactions between α-chaconine concentration and incubation time. α-Chaconine significantly reduced the relative and mRNA expressions of genes coding tight junction proteins zonula occludens-1 (ZO-1) and occludin, increased malondialdehyde (MDA) content, decreased total glutathione (T-GSH) content, reduced the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and γ-glutamylcysteine synthetase (γ-GCS) and the mRNA expressions of SOD, CAT, GSH-Px, and γ-GCS genes. In conclusion, α-chaconine disrupts the cell cycle, destroys the mechanical barrier and permeability of mucosal epithelium, inhibits cell proliferation, and accelerates cell apoptosis.

Up-Regulating ERIC by CRISPR-dCas9-VPR Inhibits Cell Proliferation and Invasion and Promotes Apoptosis in Human Bladder Cancer.

LncRNAs are defined as non-coding RNAs that are longer than 200 nucleotides in length. The previous studys has shown that lncRNAs played important roles in the regulation of gene expression and were essential in mammalian development and disease processes. Inspired by the observation that lncRNAs are aberrantly expressed in tumors, we extracted RNA from Bladder urothelial carcinoma and matched histologically normal urothelium from each patient and bladder carcinoma cell lines. Then, we reversed transcribed them into cDNA.Last, we investigated the expression patterns of ERIC by the fluorescence quantitative PCR in bladder cancer tissues and cell lines. CRISPR-dCas9-VPR targeting ERIC plasmid was transfected into T24 and 5637 cells, and cells were classified into two groups: negative control (NC) and ERIC overexpression group. MTT assay, transwell assay, and flow cytometry were performed to examine changes in cell proliferation, invasiveness, and apoptosis. We found that the expression of ERIC was down-regulated in bladder urothelial carcinoma compared to matched histologically normal urotheliam. The differences of the expression of this gene were large in the bladder cancer lines. Compared with the negative control group, the ERIC overexpression group showed significantly decreased cell proliferation rate (t = 7.583, p = 0.002; t = 3.283, p = 0.03) and invasiveness (t = 11.538, p < 0.001; t = 8.205, p = 0.01); and increased apoptotic rate (t = −34.083, p < 0.001; t = −14.316, p < 0.001). Our study lays a foundation for further study of its pathogenic mechanism in bladder cancer.

Tissue distribution of polystyrene nanoplastics in mice and their entry, transport, and cytotoxicity to GES-1cells.

With the widespread use of plastics and nanotechnology products, nanoplastics (NPs) have become a potential threat to human health. It is of great practical significance to study and evaluate the distribution of NPs in mice as mammal models and their entry, transport, and cytotoxicity in human cell lines. In this study, we detected the tissue distribution of fluorescent polystyrene nanoplastics (PS-NPs) in mice and assessed their endocytosis, transport pathways, and cytotoxic effects in GES-1cells. We found that PS-NPs were clearly visible in gastric, intestine, and liver tissues of mice and in GES-1cells treated with PS-NPs. Entry of PS-NPs into GES-1cells decreased with the inhibition of caveolae-mediated endocytosis (nystatin), clathrin-mediated endocytosis (chlorpromazine HCl), micropinocytosis (ethyl-isopropyl amiloride), RhoA (CCG-1423), and F-actin polymerization (lantrunculin A). Rac1 inhibitors (NSC 23766) had no significant effect on PS-NPs entering GES-1cells. F-actin levels significantly decreased in CCG-1423-pretreated GES-1cells exposed to PS-NPs. GES-1cell ultrastructural features indicated that internalized PS-NPs can be encapsulated in vesicles, autophagosomes, lysosomes, and lysosomal residues. RhoA, F-actin, RAB7, and LAMP1 levels in PS-NPs-treated GES-1cells were remarkably up-regulated and the Rab5 level was significantly down-regulated compared to levels in untreated cells. PS-NPs treatment decreased cell proliferation rates and increased cell apoptosis. The formation of autophagosomes and autolysosomes and levels of LC3II increased with the length of PS-NPs treatment. The results indicated that cells regulated endocytosis in response to PS-NPs through the RhoA/F-actin signaling pathway and internalized PS-NPs in the cytoplasm, autophagosomes, or lysosomes produced cytotoxicity. These results illustrate the potential threat of NPs pollution to human health.

Human adenylate kinase 6 regulates WNK1 (with no lysine kinase-1) phosphorylation states and affects ion homeostasis in NT2 cells

Adenylate kinase 6 (AK6), a nucleus localized phosphotransferase in mammalians, shows ubiquitously expression and broad substrate activity in different tissues and cell types. Although the function of AK6 has been extensively studied in different cancer cell lines, its role in mammalian germline is still unknown. Here we showed that knockdown of AK6 inhibits cell proliferation and promotes cell apoptosis in human testicular carcinoma (NT2 cells). Co-immunoprecipitation experiment and in vitro pull down assay identified WNK1 (with no lysine kinase-1) as one of the AK6 interacting proteins in NT2 cells. Moreover, we found that AK6 regulates the phosphorylation states of WNK1 (Thr60) and affects phosphorylation level of Akt (Ser473) upon hypotonic condition, probably affecting chloride channel and regulating ion transport and homeostasis in NT2 cells and consequently contributing to the decreased cell proliferation rate. In conclusion, AK6 regulates WNK1 phosphorylation states and affects ion homeostasis in NT2 cells. These findings provide new insights into the function of AK6 and WNK1 in human testicular carcinoma. This work also provides foundation for further mechanism study of AK6 in spermatogenesis.

Effects of targeted Notch1 silencing on the biological processes of the T24 and 5637 cells in vitro.

The present study aimed to investigate the roles of Notch1 in the biological processes of bladder cancer cells (BCCs) in vitro. Short hairpin (sh)RNA targeting Notch1 was designed and constructed, and the T24 and 5637 BCCs were selected for transfection. The cells were classified into two groups: shRNA negative control (NC) and Notch1 shRNA. MTT and Transwell assays, and flow cytometry were performed to examine the changes in cell proliferation, invasiveness, and apoptosis, respectively. In addition, reverse transcription-quantitative PCR and western blot analysis was used to detect the mRNA and protein expression levels of apoptosis-related proteins (Bax, Bid and Bcl2) and epithelial-mesenchymal transition factors (vimentin and E- and N-cadherin). Compared with that in the shRNA NC group, the Notch1 shRNA group showed significantly decreased cell proliferation rate and invasiveness; increased apoptotic rate; elevated mRNA expression levels of Bad, Bid and E-cadherin; and reduced mRNA expression levels of Bcl2, N-cadherin and vimentin. The trends for protein expression levels were the same as those for mRNA levels. Notch1 silencing inhibited invasion and promoted apoptosis of BCCs.

ELABELA alleviates syncytiotrophoblast hypoxia/reoxygenation injury and preeclampsia-like symptoms in mice by reducing apoptosis

IntroductionPreeclampsia (PE) is associated with increased syncytiotrophoblast apoptosis. ELABELA (ELA) is a circulating hormone secreted by the placenta. Here, we investigated the involvement of ELA in the pathogenesis of PE. MethodsWe measured ELA expression in the placental villi of patients with severe PE and healthy controls. A cellular model of hypoxia and reoxygenation was used to simulate PE hypoxia, and changes in the proliferation and apoptosis of trophoblasts in response to different ELA concentrations were measured. In addition, we used NG-nitro-l-arginine methyl ester (l-NAME) to generate a mouse model of pregnancy-induced hypertension and explore whether ELA can improve the symptoms of PE. ResultsELA expression was decreased in severe PE. ELA promoted the proliferation of BeWo cells and improved the decreased cell proliferation rate after hypoxia/reoxygenation injury. ELA reversed the phenotypes of l-NAME-induced PE mice and regulated the expression of mouse placental apoptosis factors. DiscussionELA reduced apoptosis in BeWo cells and improved PE-like symptoms in mice, suggesting its value as a potential novel treatment for PE.

Impact of Tranexamic Acid on Chondrocytes and Osteogenically Differentiated Human Mesenchymal Stromal Cells (hMSCs) In Vitro.

The topical application of tranexamic acid (TXA) helps to prevent post-operative blood loss in total joint replacements. Despite these findings, the effects on articular and periarticular tissues remain unclear. Therefore, this in vitro study examined the effects of varying exposure times and concentrations of TXA on proliferation rates, gene expression and differentiation capacity of chondrocytes and human mesenchymal stromal cells (hMSCs), which underwent osteogenic differentiation. Chondrocytes and hMSCs were isolated and multiplied in monolayer cell cultures. Osteogenic differentiation of hMSCs was induced for 21 days using a differentiation medium containing specific growth factors. Cell proliferation was analyzed using ATP assays. Effects of TXA on cell morphology were examined via light microscopy and histological staining, while expression levels of tissue-specific genes were measured using semiquantitative RT-PCR. After treatment with 50 mg/mL of TXA, a decrease in cell proliferation rates was observed. Furthermore, treatment with concentrations of 20 mg/mL of TXA for at least 48 h led to a visible detachment of chondrocytes. TXA treatment with 50 mg/mL for at least 24 h led to a decrease in the expression of specific marker genes in chondrocytes and osteogenically differentiated hMSCs. No significant effects were observed for concentrations beyond 20 mg/mL of TXA combined with exposure times of less than 24 h. This might therefore represent a safe limit for topical application in vivo. Further research regarding in vivo conditions and effects on hMSC functionality are necessary to fully determine the effects of TXA on articular and periarticular tissues.

Association between vitamin D and ovarian cancer development in BRCA1 mutation carriers.

Objective: Women with inherited mutations in BRCA1 gene have a high (40–70%) genetic risk of developing ovarian cancer. Epidemiological studies suggest an inverse correlation between serum vitamin D (VD) levels and the risk of ovarian cancer, but there is a lack of data from BRCA1 mutation (BRCA1mut) carriers. Therefore, we investigated VD levels and actions in cancer free women with BRCA1 mutations.Materials and Methods: Blood, ovary and fallopian tube samples were collected from healthy pre-menopausal women with BRCA1mut and without BRCA1 mutations (BRCAwt). Serum calcifediol (major circulating form of VD) concentrations were measured by electrochemiluminescence immunoassay. Immunohistochemistry was performed on paraffin-embedded ovarian and fallopian tube sections to determine vitamin D receptor (VDR) expression. Ovarian surface epithelial cells (OSEs) from BRCA1mut carriers were cultured with or without calcitriol supplementation for 72 hrs. VDR protein levels, cell proliferation and cell viability were analyzed.Results: BRCA1mut women had lower serum calcifediol levels compared to BRCAwt women (p = 0.003). VDR protein expression was evident in ovarian and the fallopian tube epithelium of BRCAwt patients, but was reduced in BRCA1mut women. Calcitriol (biologically active VD) supplementation elevated VDR expression in cultured BRCA1mut OSEs (p = 0.005) and decreased cell proliferation rates in a dose-dependent manner without inducing apoptosis.Conclusions: VD biosynthesis and signaling via VDR in the ovarian and fallopian tube epithelium are impaired in BRCA1mut women. VD treatment may limit BRCA1mut epithelial cell proliferation without affecting cell viability, providing a rationale for exploring the potential for VD in ovarian cancer prevention in BRCA1mut carriers.