Exploring the anti-atherosclerosis mechanism of ginsenoside Rb1 by integrating network pharmacology and experimental verification.

Ginsenoside Rb1 is the major active constituent of ginseng, which is widely used in traditional Chinese medicine for the atherosclerosis treatment by anti-inflammatory, anti-oxidant and reducing lipid accumulation. We explored cellular target and molecular mechanisms of ginsenoside Rb1 based on network pharmacology and in vitro experimental validation. In this study, we predicted 17 potential therapeutic targets for ginsenoside Rb1 with atherosclerosis from public databases. We then used protein-protein interaction network to screen the hub targets. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment showed that the effects of ginsenoside Rb1 were meditated through multiple targets and pathways. Next, molecular docking results revealed that in the 10 core targets, CCND1 has the highest binding energy with ginsenoside Rb1. Vascular cell proliferation plays a critical role in atherosclerosis development. However, the effect and direct target of ginsenoside Rb1 in regulating vascular cell proliferation in atherosclerosis remains unclear. Edu straining results indicated that ginsenoside Rb1 inhibited the cell proliferation of endothelial cells, macrophages, and vascular smooth muscle cells. The protein immunoprecipitation (IP) analysis showed that ginsenoside Rb1 inhibited the vascular cell proliferation by suppressing the interaction of CCDN1 and CDK4. These findings systematically reveal that the anti-atherosclerosis mechanism of ginsenoside Rb1 by integrating network pharmacology and experimental validation, which provide evidence to treat atherosclerosis by using ginsenoside Rb1 and targeting CCND1.

Comparison of phenolics, antioxidant, and antiproliferative activities of two Hypsizygus marmoreus varieties.

Phenolics, antioxidant activities, and antiproliferative properties of brown Hypsizygus marmoreus (brown HM) and white Hypsizygus marmoreus (white HM) were compared. The results showed that the contents of (+)-catechin, gallic acid, and protocatechuic acid of brown HM were higher than those of white HM. Moreover, brown HM had greater cellular antioxidant activity (CAA), peroxyl radical scavenging capacity (PSC), and oxygen radical absorbance capacity (ORAC) values than white HM, which demonstrated that brown HM presented a stronger antioxidant capacity. Both of brown HM and white HM showed remarkable antiproliferative activities against HepG2 cells and brown HM was proven to be the more effective. The flow cytometry results revealed that both of brown HM and white HM could induce G1 arrest and cell apoptotics in a dose-dependent manner. In addition, CyclinD1, CDK4, and Bcl-2 mRNA expression levels were downregulated with the treatment of brown HM or white HM. Taken together, our study revealed that brown HM afforded better antioxidant and antiproliferative activities than white HM and laid the foundation for potential application of Hypsizygus marmoreus as source of nutraceuticals and functional food products. PRACTICAL APPLICATION: A systematic assessment of the potential differences of phenolics, antioxidant, and antiproliferative activities between different Hypsizygus marmoreus varieties was carried out in the present study. Furthermore, our findings would present possible antiproliferative mechanism of extracts of different Hypsizygus marmoreus varieties, which may provide theoretical basis for further development and utilization of Hypsizygus marmoreus.

ß-Thujaplicin inhibits basal-like mammary tumor growth by regulating glycogen synthase kinase-3ß/ß-catenin signaling.

ß-Thujaplicin, a natural monoterpenoid, has been demonstrated to exert health beneficial activities in chronic diseases. However, it has not been studied in regulating estrogen receptor (ER) negative breast cancer. Here, we investigated the effect of ß-thujaplicin on inhibiting ER-negative basal-like breast cancer and the underlying mechanism of action using an in vitro and in vivo xenograft animal model. ß-Thujaplicin induced G0/G1 phase cell cycle arrest and regulated cell cycle mediators, cyclin D1, cyclin E, and cyclin-dependent kinase 4 (CDK 4), leading to the inhibition of the proliferation of ER-negative basal-like MCF10DCIS.com human breast cancer cells. It also modulated the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase (GSK-3ß) and the protein level of ß-catenin. In an MCF10DCIS.com xenograft animal model, ß-thujaplicin significantly inhibited tumor growth, reduced tumor weight, and regulated the expression of cell cycle proteins, phosphorylation of AKT and GSK-3ß, and protein level of ß-catenin in the tumor tissues. These results demonstrate that ß-thujaplicin can suppress basal-like mammary tumor growth by regulating GSK-3ß/ß-catenin signaling, suggesting that ß-thujaplicin may be a potent chemopreventive agent against the basal-like subtype of breast cancer.

Lipophilic Grape Seed Proanthocyanidin Exerts Anti-Proliferative and Pro-Apoptotic Effects on PC3 Human Prostate Cancer Cells and Suppresses PC3 Xenograft Tumor Growth in Vivo.

The in vitro antiprostate cancer activity of lipophilic grape seed proanthocyanidin (LGSP) against the PC3 cell line was evaluated by MTT assay, flow cytometry, and immunoblot analysis, and the in vivo antiprostate cancer effect was evaluated by a PC3-derived mouse xenograft model via oral gavage LGSP. Ki67 and cleaved caspase 3 immunostaining experiments were performed in tumor tissues. LGSP exhibited a strong inhibitory effect on PC3 cell proliferation by inducing apoptosis. Treatment with LGSP resulted in a G1 phase cell cycle arrest in PC3 cells, which was further confirmed by decreasing the expression of cyclin D1 and CDK 4 and increasing the expression of the tumor suppressors p21 and p27. Furthermore, activation of cleaved fragments of caspases 3, caspases 9, and PARP indicated that LGSP-induced apoptosis is caspase-dependent. Upstream of caspase cascade, LGSP increased the cytochrome c release in cytoplasm. After treatment with LGSP, the Bcl-2/Bax ratio also decreased in PC3 cells. In tumor studies, LGSP inhibited the growth of PC3-derived mouse xenografts by inhibiting tumor cell proliferation and inducing apoptosis. Our findings suggest that LGSP is an effective antiprostate cancer component and deserves further study.

Guanidinoacetic Acid Regulates Myogenic Differentiation and Muscle Growth Through miR-133a-3p and miR-1a-3p Co-mediated Akt/mTOR/S6K Signaling Pathway.

Guanidinoacetic acid (GAA), an amino acid derivative that is endogenous to animal tissues including muscle and nerve, has been reported to enhance muscular performance. MicroRNA (miRNA) is a post-transcriptional regulator that plays a key role in nutrient-mediated myogenesis. However, the effects of GAA on myogenic differentiation and skeletal muscle growth, and the potential regulatory mechanisms of miRNA in these processes have not been elucidated. In this study, we investigated the effects of GAA on proliferation, differentiation, and growth in C2C12 cells and mice. The results showed that GAA markedly inhibited the proliferation of myoblasts, along with the down-regulation of cyclin D1 (CCND1) and cyclin dependent kinase 4 (CDK4) mRNA expression, and the upregulation of cyclin dependent kinase inhibitor 1A (P21) mRNA expression. We also demonstrated that GAA treatment stimulated myogenic differentiation 1 (MyoD) and myogenin (MyoG) mRNA expression, resulting in an increase in the myotube fusion rate. Meanwhile, GAA supplementation promoted myotube growth through increase in total myosin heavy chain (MyHC) protein level, myotubes thickness and gastrocnemius muscle cross-sectional area. Furthermore, small RNA sequencing revealed that a total of eight miRNAs, including miR-133a-3p and miR-1a-3p cluster, showed differential expression after GAA supplementation. To further study the function of miR-133a-3p and miR-1a-3p in GAA-induced skeletal muscle growth, we transfected miR-133a-3p and miR-1a-3p mimics into myotube, which also induced muscle growth. Through bioinformatics and a dual-luciferase reporter system, the target genes of miR-133a-3p and miR-1a-3p were determined. These two miRNAs were shown to modulate the Akt/mTOR/S6K signaling pathway by restraining target gene expression. Taken together, these findings suggest that GAA supplementation can promote myoblast differentiation and skeletal muscle growth through miR-133a-3p- and miR-1a-3p-induced activation of the AKT/mTOR/S6K signaling pathway.

Schisantherin A Improves Learning and Memory of Mice with D-Galactose-Induced Learning and Memory Impairment Through Its Antioxidation and Regulation of p19/p53/p21/Cyclin D1/CDK4/RB Gene Expressions.

Schisantherin A (SCA) was evaluated for possible function in restoring the learning and memory impairment induced by D-galactose in mice. ICR mice were treated with D-galactose subcutaneously (220 mg·kg-1), and followed by SCA in different doses (1.25, 2.50 and 5.00 mg·kg-1, administered orally) for 42 days. Effects of SCA on learning and memory were examined by step-through tests and Morris water maze tests. The activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA) in the peripheral blood and hippocampus of mice were assayed by water-soluble tetrazolium-1 (WST-1) and thiobarbituric acid (TBA) methods. The contents of 8 hydroxy deoxy guanosine (8-OHdG) in the hippocampus of mice were detected by immunosorbent assay methods, respectively. Quantitative real-time PCR and Western Blot were respectively used to detect the expression of p19, p53, p21, cyclin D1, CDK4 and RB genes, and the phosphorylation of RB in the hippocampus of mice. We found that SCA significantly improved the learning and memory impairment induced by D-galactose in mice. After SCA treatment, SOD activity was increased and the content of MDA was decreased in both peripheral blood and hippocampus of mice. 8-OHDG content was also decreased in the hippocampus of mice. Furthermore, the expression of p19, p53 and p21 genes was reduced and the expression of cyclin D1 and CDK4 and the phosphorylation of RB protein were elevated in the hippocampus. SCA may improve the learning and memory impairment induced by D-galactose by enhancing the antioxidant capacity, and regulating the expression of p19/p53/p21/cyclinD1/CDK4 genes, and the phosphorylation of RB protein in the hippocampus of mice.

Vitex rotundifolia Fruit Suppresses the Proliferation of Human Colorectal Cancer Cells through Down-regulation of Cyclin D1 and CDK4 via Proteasomal-Dependent Degradation and Transcriptional Inhibition.

Viticis Fructus (VF) as the dried fruit from Vitex rotundifolia L. used as a traditional medicine for treating inflammation, headache, migraine, chronic bronchitis, eye pain, and gastrointestinal infections has been reported to have antiproliferative effects against various cancer cells, including breast, lung and colorectal cancer cells. However, the molecular mechanisms by which VF mediates the inhibitory effect of the proliferation of cancer cells have not been elucidated in detail. In this study, we investigated the molecular mechanism of VF on the down-regulation of cyclin D1 and CDK4 level associated with cancer cell proliferation. VF suppressed the proliferation of human colorectal cancer cell lines such as HCT116 and SW480. VF induced decrease in cyclin D1 and CDK4 in both protein and mRNA levels. However, the protein levels of cyclin D1 and CDK4 were decreased by VF at an earlier time than the change of mRNA levels; rather it suppressed the expression of cyclin D1 and CDK4 via the proteasomal degradation. In cyclin D1 and CDK4 degradation, we found that Thr286 phosphorylation of cyclin D1 plays a pivotal role in VF-mediated cyclin D1 degradation. Subsequent experiments with several kinase inhibitors suggest that VF-mediated degradation of cyclin D1 may be dependent on GSK3[Formula: see text] and VF-mediated degradation of CDK4 is dependent on ERK1/2, p38 and GSK3[Formula: see text]. In the transcriptional regulation of cyclin D1 and CDK4, we found that VF inhibited Wnt activation associated with cyclin D1 transcriptional regulation through TCF4 down-regulation. In addition, VF treatment down-regulated c-myc expression associated CDK4 transcriptional regulation. Our results suggest that VF has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Antiproliferative Effects of a Triterpene-Enriched Extract from Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), on Human Lung Cancer Cells.

Ganoderma lucidum, a mushroom that has been used to treat disease in East Asia for centuries, has been shown to be effective against many types of tumors, but the exact cellular mechanism of action is unknown. In this study we examined proliferation of a lung cancer cell line after treatment with 12 concentrations of powdered G. lucidum for 24, 48, and 120 hours. Based on half-maximal inhibitory concentrations values, proliferation of the H1793 cell line seemed to be sensitive to the extract in a time- and dose-dependent manner. We used immunoblot analysis to examine the amounts of cell cycle proteins (cyclin D, Cdk4, and Cdc2) and apoptotic proteins (Bcl-xL and Bax) after treatment with a range of G. lucidum concentrations. Changes in amounts of proteins that regulate the cell cycle were consistent with longer G1 and G2 phases. Proapoptotic protein (Bax) levels increased 6.5-fold, with a commensurate increase in the Bax-to-Bcl ratio, especially at 48 and 120 hours. These results suggest that the decrease in cellular proliferation correlated with a change in both cell cycle progression and apoptosis, and that the triterpenoid in G. lucidum is the bioactive component. Further biochemical characterization of this ancient herbal remedy could hold promise for treating lung cancer.

Effect of single nucleotide polymorphisms on CDK4 and Zn supplementation in children with growth hormone deficiency.

OBJECTIVE: Precision medicine is a personalized disease prevention and treatment program combining modern genetic technology, molecular imaging techniques, and biological information with patients' living environment and clinical data, for accurate classification and diagnosis of diseases. CASE REPORT: Our study presents the case of a 7-year-old female patient with clinical manifestations of growth hormone (GH) deficiency. After treatment with recombinant human GH for 2 years, the patient showed a reduced growth rate. Then single nucleotide polymorphisms according to GHD was analyzed, and the 6009 site within cyclin-dependent kinase 4 gene showed a weak response to IGF-1 which was a downstream signal molecules of GH. CONCLUSIONS: Based on these results, both Zn and GH were supplied to the patients, and the growth rate increased significantly. Precision medicine needs more studies on patients to make accurate treatment.

Secoisolariciresinol diglucoside rich extract of L. usitatissimum prevents diabetic colon cancer through inhibition of CDK4.

BACKGROUND: There is increased risk of colon cancer in both men and women having diabetes. The objective of the study was to evaluate the role of Secoisolariciresinol diglucoside rich extract(SRE) of L.usissatisimum(flaxseed) in colon cancer associated with type 2 diabetes mellitus. MATERIAL AND METHODS: Diabetes was induced by administering high fat diet with low dose streptozotocin model. After 6 weeks, diabetes was confirmed and 1,2 dimethylhydrazine(25mg/kg, sc) weekly administration was from 6th to 18th weeks. Rats were treated with the SRE(500mg/kg) orally from 6th to 24th week. After 24 weeks, various biochemical and enzymatic parameters were estimated. Animals were sacrificed and colon tissue was separated and subjected to analysis of histopathological, PCNA studies and mRNA expression of CDK4. RESULTS: Disease control rats depicted hyperglycaemia, hyperinsulinaemia, elevated pro-inflammatory cytokines and cancer biomarker levels, and marked presence of proliferating cells. Treatment with SRE controlled hyperglycaemia, hyperinsulinaemia, reduced pro-inflammatory cytokines and cancer biomarker levels, and decreased no. of proliferating cells. We found that disease control rats depicted over expression of CDK4 mRNA levels which were reduced by SRE treatment. CONCLUSIONS: SRE of L. usitatissimum exhibited chemopreventive effect in colon cancer associated with type 2 diabetes mellitus which might be mediated through inhibition of CDK4.

Coptis japonica Makino extract suppresses angiogenesis through regulation of cell cycle-related proteins.

Angiogenesis, neovascularization from pre-existing vessels, is a key step in tumor growth and metastasis, and anti-angiogenic agents that can interfere with these essential steps of cancer development are a promising strategy for human cancer treatment. In this study, we characterized the anti-angiogenic effects of Coptis japonica Makino extract (CJME) and its mechanism of action. CJME significantly inhibited the proliferation, migration, and invasion of vascular endothelial growth factor (VEGF)-stimulated HUVECs. Furthermore, CJME suppressed VEGF-induced tube formation in vitro and VEGF-induced microvessel sprouting ex vivo. According to our study, CJME blocked VEGF-induced cell cycle transition in G1. CJME decreased expression of cell cycle-regulated proteins, including Cyclin D, Cyclin E, Cdk2, and Cdk4 in response to VEGF. Taken together, the results of our study indicate that CJME suppresses VEGF-induced angiogenic events such as proliferation, migration, and tube formation via cell cycle arrest in G1.

Effect of Fanbaicao (Herba Potentillae Discoloris) oil on the expression of p21 and CDK4 in HepG2 cells.

OBJECTIVE: To research the anti-cancer mechanism of the Traditional Chinese Medicine Fanbaicao (Herba Potentillae Discoloris) oil in the human hepatoma cell line HepG2. METHODS: Gas chromatography was used to analyze the components of Fanbaicao (Herba Potentillae Discoloris). We tested the inhibitory effect of Fanbaicao (Herba Potentillae Discoloris) oil on the human hepatoma cell line HepG2 in vitro using 3-(4, 5-Dimet hylt hiazol-2-yl)-2,5-dip henyltetrazolium bromide assays. Fluorescence activating cell sorter analysis was used to examine the levels of apoptosis, and western blot and immunofluorescence were used to detect the expression of p21, p-p21 and CDK4 proteins. RESULTS: Fanbaicao (Herba Potentillae Discoloris) oil contains 45 ingredients, and L-ascorbic acid 2, 6-bispalmitate was the main component and accounted for 44.96% of total drive-off peak area. Other components included (Z)-14-met hyl-8-exadecenal- acetal (8.56%), phytol (7.74%) and lauric acid (6.31% ). Fanbaicao (Herba Potentillae Discoloris) oil treatment reduced the proliferation of HepG2 cells and the half growth inhibition concentration (IC50) was 2.03 mg/mL. Furthermore, we also observed significantly increased HepG2 cell apoptosis in a dose-dependent manner (P < 0.05). Fanbaicao (Herba Potentillae Discoloris) oil significantly increased the expression of p21 and p-p21 and significantly decreased the expression of CDK4 in HepG2 cells compared with controls (P < 0.01). CONCLUSION: Our results showed that Fanbaicao (Herba Potentillae Discoloris) oil has anti-cancer activities in HepG2 cells, which is probably related to the upregulation of p21 and p-p21 and downregulation of CDK4 expression.

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Analysing the Effect of Mutation on Protein Function and Discovering Potential Inhibitors of CDK4: Molecular Modelling and Dynamics Studies.

The cyclin-dependent kinase 4 (CDK4)-cyclin D1 complex plays a crucial role in the transition from the G1 phase to S phase of the cell cycle. Among the CDKs, CDK4 is one of the genes most frequently affected by somatic genetic variations that are associated with various forms of cancer. Thus, because the abnormal function of the CDK4-cyclin D1 protein complex might play a vital role in causing cancer, CDK4 can be considered a genetically validated therapeutic target. In this study, we used a systematic, integrated computational approach to identify deleterious nsSNPs and predict their effects on protein-protein (CDK4-cyclin D1) and protein-ligand (CDK4-flavopiridol) interactions. This analysis resulted in the identification of possible inhibitors of mutant CDK4 proteins that bind the conformations induced by deleterious nsSNPs. Using computational prediction methods, we identified five nsSNPs as highly deleterious: R24C, Y180H, A205T, R210P, and R246C. From molecular docking and molecular dynamic studies, we observed that these deleterious nsSNPs affected CDK4-cyclin D1 and CDK4-flavopiridol interactions. Furthermore, in a virtual screening approach, the drug 5_7_DIHYDROXY_ 2_ (3_4_5_TRI HYDROXYPHENYL) _4H_CHROMEN_ 4_ONE displayed good binding affinity for proteins with the mutations R24C or R246C, the drug diosmin displayed good binding affinity for the protein with the mutation Y180H, and the drug rutin displayed good binding affinity for proteins with the mutations A205T and R210P. Overall, this computational investigation of the CDK4 gene highlights the link between genetic variation and biological phenomena in human cancer and aids in the discovery of molecularly targeted therapies for personalized treatment.

Targeting of multiple oncogenic signaling pathways by Hsp90 inhibitor alone or in combination with berberine for treatment of colorectal cancer.

There is a wide range of drugs and combinations under investigation and/or approved over the last decade to treat colorectal cancer (CRC), but the 5-year survival rate remains poor at stages II-IV. Therefore, new, more-efficient drugs still need to be developed that will hopefully be included in first-line therapy or overcome resistance when it appears, as part of second- or third-line treatments in the near future. In this study, we revealed that heat shock protein 90 (Hsp90) inhibitors have high therapeutic potential in CRC according to combinative analysis of NCBI's Gene Expression Omnibus (GEO) repository and chemical genomic database of Connectivity Map (CMap). We found that second generation Hsp90 inhibitor, NVP-AUY922, significantly downregulated the activities of a broad spectrum of kinases involved in regulating cell growth arrest and death of NVP-AUY922-sensitive CRC cells. To overcome NVP-AUY922-induced upregulation of survivin expression which causes drug insensitivity, we found that combining berberine (BBR), a herbal medicine with potency in inhibiting survivin expression, with NVP-AUY922 resulted in synergistic antiproliferative effects for NVP-AUY922-sensitive and -insensitive CRC cells. Furthermore, we demonstrated that treatment of NVP-AUY922-insensitive CRC cells with the combination of NVP-AUY922 and BBR caused cell growth arrest through inhibiting CDK4 expression and induction of microRNA-296-5p (miR-296-5p)-mediated suppression of Pin1-ß-catenin-cyclin D1 signaling pathway. Finally, we found that the expression level of Hsp90 in tumor tissues of CRC was positively correlated with CDK4 and Pin1 expression levels. Taken together, these results indicate that combination of NVP-AUY922 and BBR therapy can inhibit multiple oncogenic signaling pathways of CRC.

Anti-proliferative effects of qianliening capsules on prostatic hyperplasia in vitro and in vivo.

Previous studies by our group showed that Qianliening capsules (QC), a clinically proven effective traditional Chinese formulation that has long been used in the treatment of benign prostatic hyperplasia (BPH), is capable of inhibiting BPH in vivo and in vitro via the promotion of apoptosis, suppression of the EGFR/STAT3 signaling pathway and regulating the expression of sex hormones as well as their receptors. However, the mechanism of its anti-BPH activity has remained to be fully elucidated. The present study aimed to investigate the mechanism underlying the anti-proliferative effect of QC in vivo and in vitro. Castrated male Sprage-Dawley (SD) rats where subcutaneously injected with testosterone propionate and the WPMY-1 cell line was stimulated with basic fibroblast growth factor in order to generate BPH in vivo and in vitro separately, both of which were then subjected to QC treatment. Finasteride was used as a positive control drug for the in vivo study. In the present study, it was found that treatment with QC or finasteride significantly reduced the prostatic index (PI=prostate wet weight/body weight x 100) in a rat model of BPH (P<0.05). In addition, reverse transcription quantitative polymerase chain reaction (RT-PCR) and western blot analyses showed that QC or finasteride treatment significantly inhibited model construction-induced upregulation of expression of proliferating cell nuclear antigen, cyclin D1 and cyclin-dependent kinase 4 in prostatic tissues of rats with BPH (P<0.05). The in vitro study further proved that QC exhibited anti-proliferative properties via G1/S cell cycle arrest in the WPMY-1 cell line, as evidenced by colony formation, flow cytometric cell cycle, immunoblot and RT-PCR analyses. In conclusion, the present study demonstrated that inhibition of cell proliferation via G1/S cell cycle arrest may be one of the underlying mechanisms of the effect of QC on BPH.

CDK4-mediated MnSOD activation and mitochondrial homeostasis in radioadaptive protection.

Mammalian cells are able to sense environmental oxidative and genotoxic conditions such as the environmental low-dose ionizing radiation (LDIR) present naturally on the earth's surface. The stressed cells then can induce a so-called radioadaptive response with an enhanced cellular homeostasis and repair capacity against subsequent similar genotoxic conditions such as a high dose radiation. Manganese superoxide dismutase (MnSOD), a primary mitochondrial antioxidant in mammals, has long been known to play a crucial role in radioadaptive protection by detoxifying O2(•-) generated by mitochondrial oxidative phosphorylation. In contrast to the well-studied mechanisms of SOD2 gene regulation, the mechanisms underlying posttranslational regulation of MnSOD for radioprotection remain to be defined. Herein, we demonstrate that cyclin D1/cyclin-dependent kinase 4 (CDK4) serves as the messenger to deliver the stress signal to mitochondria to boost mitochondrial homeostasis in human skin keratinocytes under LDIR-adaptive radioprotection. Cyclin D1/CDK4 relocates to mitochondria at the same time as MnSOD enzymatic activation peaks without significant changes in total MnSOD protein level. The mitochondrial-localized CDK4 directly phosphorylates MnSOD at serine-106 (S106), causing enhanced MnSOD enzymatic activity and mitochondrial respiration. Expression of mitochondria-targeted dominant negative CDK4 or the MnSOD-S106 mutant reverses LDIR-induced mitochondrial enhancement and adaptive protection. The CDK4-mediated MnSOD activation and mitochondrial metabolism boost are also detected in skin tissues of mice receiving in vivo whole-body LDIR. These results demonstrate a unique CDK4-mediated mitochondrial communication that allows cells to sense environmental genotoxic stress and boost mitochondrial homeostasis by enhancing phosphorylation and activation of MnSOD.

[Effect of Buyang Huanwu decoction and its simple prescription (Naojian tablet) on CDK4/Cyclin D1 expression of rats with cerebral ischemia].

To evaluate the regulating effect of Buyang Huanwu decoction and its simple prescription (Naojian tablet) on CDK4/Cyclin D1 expression in hippocampus tissues of rats with cerebral ischemia, SD rats were divided into the sham-operation group, the model group, the Buyang Huanwu decoction group (ig, 3.15 g · kg⁻¹) and the simple prescription group (ig, 2.41 g · kg⁻¹). Each group was further divided into five subgroups based on time points after the administration, i. e. 1 d, 3 d, 7 d, 14 d and 28 d, respectively. CDK4/Cyclin D1 expressions of the group at different time points were examined by using immunohistochemistry and real-time qPCR. According to the results, the cerebral ischemia model group showed higher CDK4/Cyclin D1 expression than the sham-operation groups (P < 0.05), suggesting that the cell cycle signal pathway would be activated by the cerebral ischemic injury. Both Buyang Huanwu decoction and simple prescription groups showed significantly lower cyclin expression than the model group at 3 d, 7 d, 14 d, 28 d (P < 0.05), indicating both Buyang Huanwu decoction and its simple prescription could play the neuroprotective effect through the cell cycle signal pathway.

Tougu Xiaotong formula induces chondrogenic differentiation in association with transforming growth factor-ß1 and promotes proliferation in bone marrow stromal cells.

Indian hedgehog (Ihh), one of the hedgehog gene families, is indicated in the regulation of chondrocyte differentiation. Tougu Xiaotong formula (TXF), a traditional Chinese medicinal compound, has been used for the treatment of bone and joint disease. However, the underlying molecular mechanisms of TXF on the function of bone marrow stromal cells (BMSCs) remain unclear. In the present study, the affect of TXF on proliferation and chondrogenic differentiation was investigated in primary BMSCs from four­week­old Sprague Dawley rats. The cell viability in BMSCs treated with TXF was higher compared to the untreated cells. Additionally, the percentage of G(0)/G(1) phase cells was significantly decreased, whereas that of the S phase cells was significantly increased. Furthermore, following TXF treatment, cyclin D1, cyclin­dependent kinase 4 (CDK4) and CDK6 expression in BMSCs was significantly enhanced. The results showed that TXF had no cytotoxicity to BMSCs. To explore the effect of TXF on the differentiation in BMSCs, whether TXF induced chondrogenic differentiation of BMSCs by the regulation of Ihh signaling pathway was investigated. The protein expression of Ihh, Patched and Smoothened in the induction group were significantly increased when compared to those in the control group, and the highest protein level of Ihh was in the induction group that was treated with the combination of TXF and transforming growth factor­ß1 (TGF­ß1). In addition, TXF combined with TGF­ß1 significantly induced the protein expression of cartilage oligomeric matrix protein and collagen II compared to the TGF­ß1 group. Taken together, these results indicate that TXF promotes the proliferation via accelerating the G(1)/S transition, and induces chondrogenic differentiation in BMSCs by activation of the Ihh signaling pathway in association with TGF­ß1.

Magnolol causes alterations in the cell cycle in androgen insensitive human prostate cancer cells in vitro by affecting expression of key cell cycle regulatory proteins.

Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 µM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16(INK4a), p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.