Molecular mechanisms of Codonopsis pilosula in inhibiting hepatocellular carcinoma growth and metastasis.

BACKGROUND: Liver cancer, one of the most common types of cancer worldwide, accounts for millions of cases annually. With its multi-target and wide-ranging therapeutic effects, traditional Chinese medicine has emerged as a potential approach for treating various tumors. Codonopsis pilosula, a traditional herb, is known for its anti-inflammatory and antioxidant properties. In this study, we investigated the potential molecular mechanisms of Codonopsis pilosula in regulating the inhibition of CDK1 and the modulation of PDK1/ß-catenin, which are involved in hepatocellular carcinoma growth and metastasis. STUDY DESIGN/METHODS: Firstly, we screened the active chemical constituents of Codonopsis pilosula and identified their respective target proteins using the Herb database. Then, we applied the GeneCards database and transcriptome sequencing analysis to screen for critical genes associated with the occurrence and development of liver cancer. The intersection of the target proteins and disease-related genes was used to determine the potential targets of Codonopsis pilosula in hepatocellular carcinoma. Protein-protein interaction analysis and GO/KEGG analysis were subsequently performed to uncover the pathways through which Codonopsis pilosula acts on liver cancer. The Huh-7 cell line, exhibiting the highest sensitivity to Codonopsis pilosula polysaccharide solution (CPP) intervention, was chosen for subsequent studies. Cell viability was evaluated using the CCK-8 assay, colony formation assay was conducted to determine cell proliferation capacity, flow cytometry was used to analyze cell cycle, TUNEL staining was performed to assess cell apoptosis, scratch assay was carried out to evaluate cell migration ability, the expression of EMT-related proteins was detected and analyzed, and cell sphere formation assay was conducted to investigate cell stemness. Finally, a liver cancer animal model was established, and different doses of CPP were administered via gavage the next day. The expression levels of CDK1, PDK1, and ß-catenin in mouse liver tissues were detected and analyzed, immunohistochemistry staining was performed to assess the expression of tumor cell proliferation-related proteins Ki67 and PCNA in mouse xenografts, and TUNEL staining was carried out to evaluate cell apoptosis in mouse liver tissues. After intervention with CDK1 expression, the expression levels of CDK1, PDK1, and ß-catenin proteins and mRNA in each group of cells were detected using Western blot and RT-qPCR. RESULTS: Through network pharmacology analysis, transcriptome sequencing, and bioinformatics analysis, 35 target genes through which Codonopsis pilosula acts on liver cancer were identified. Among them, CDK1, with the highest degree in the PPI network, was considered an essential target protein for Codonopsis pilosula in treating liver cancer. In vitro cell experiments revealed that CPP could inhibit the expression of CDK1/PDK1/ß-catenin signaling axis factors, suppress cell proliferation, decrease cell migration ability, influence the EMT process, and reduce cell stemness by inhibiting CDK1 and affecting the PDK1/ß-catenin signaling axis. Similarly, in vivo experiments demonstrated that CPP could regulate the CDK1/PDK1/ß-catenin signaling axis, inhibit tumor growth, and induce cell apoptosis. CONCLUSION: Codonopsis pilosula may inhibit hepatocellular carcinoma growth by suppressing CDK1 and affecting the PDK1/ß-catenin signaling axis, limiting cell EMT and reducing cell stemness. These findings provide insights into the potential therapeutic role of Codonopsis pilosula in liver cancer.

Comprehensive multi-omics analysis reveals WEE1 as a synergistic lethal target with hyperthermia through CDK1 super-activation.

Hyperthermic intraperitoneal chemotherapy's role in ovarian cancer remains controversial, hindered by limited understanding of hyperthermia-induced tumor cellular changes. This limits developing potent combinatory strategies anchored in hyperthermic intraperitoneal therapy (HIPET). Here, we perform a comprehensive multi-omics study on ovarian cancer cells under hyperthermia, unveiling a distinct molecular panorama, primarily characterized by rapid protein phosphorylation changes. Based on the phospho-signature, we pinpoint CDK1 kinase is hyperactivated during hyperthermia, influencing the global signaling landscape. We observe dynamic, reversible CDK1 activity, causing replication arrest and early mitotic entry post-hyperthermia. Subsequent drug screening shows WEE1 inhibition synergistically destroys cancer cells with hyperthermia. An in-house developed miniaturized device confirms hyperthermia and WEE1 inhibitor combination significantly reduces tumors in vivo. These findings offer additional insights into HIPET, detailing molecular mechanisms of hyperthermia and identifying precise drug combinations for targeted treatment. This research propels the concept of precise hyperthermic intraperitoneal therapy, highlighting its potential against ovarian cancer.

Identification of the molecular targets and mechanisms of compound mylabris capsules for hepatocellular carcinoma treatment through network pharmacology and bioinformatics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese herbal formulas have been proven to exert an inhibitory effect on tumor. Compound mylabris capsules (CMC) has been used for treating cancer, especially hepatocellular carcinoma (HCC), for years in China. However, its therapeutic mechanisms on HCC remain unclear. AIM OF THE STUDY: This research aimed to elucidate the molecular targets and mechanisms of CMC for treating HCC. MATERIALS AND METHODS: First, the bioactive ingredients and potential targets of CMC, as well as HCC-related targets were retrieved from publicly available databases. Next, the overlapped genes between potential targets of CMC and HCC-related targets were determined using bioinformatics analysis. Then, networks of ingredient-target and gene-pathway were constructed. Finally, cell experiments were carried out to examine the effects of CMC-medicated serum on HCC and validate the core molecular targets. RESULTS: In total, 151 bioactive ingredients and 255 potential targets of CMC were selected, 982 differentially expressed genes of HCC were identified. Among them, 34 overlapped genes were finally selected. In addition, 20 pathways and 429 GO terms were significantly enriched. Protein-protein interaction and gene-pathway networks indicated that Cyclin B1(CCNB1) and Cyclin Dependent Kinase 1(CDK1) were the core gene targets for the treatment of CMC on HCC. Moreover, in vitro studies showed that CMC-medicated serum significantly inhibited the viability of HepG2 cells. Furthermore, CMC downregulated CCNB1 and CDK1 expressions and induced G2/M phase cell cycle arrest. CONCLUSIONS: CMC plays a therapeutic role in HCC via multi-component, -target and -pathway mechanisms, in which CCNB1 and CDK1 may be the core molecular targets. This study indicates that the integration of network pharmacology and bioinformatics analysis, followed by experimental validation, can serves as an effective tool for studying the therapeutic mechanisms of traditional Chinese medicine.

Dracocephalum heterophyllum (DH) Exhibits Potent Anti-Proliferative Effects on Autoreactive CD4+ T Cells and Ameliorates the Development of Experimental Autoimmune Uveitis.

Experimental autoimmune uveitis (EAU) is a CD4+ T cell-mediated organ-specific autoimmune disease and has been considered as a model of human autoimmune uveitis. Dracocephalum heterophyllum (DH) is a Chinese herbal medicine used in treating hepatitis. DH suppressed the production of inflammatory cytokines through the recruitment of myeloid-derived suppressor cells (MDSCs) to the liver. However, it remains elusive whether DH can directly regulate CD4+ T cell biology and hence ameliorates the development of CD4+ T cell-mediated autoimmune disease. In the current study, we found that DH extract significantly suppressed the production of pro-inflammatory cytokines by CD4+ T cells. Further study showed that DH didn't affect the activation, differentiation, and apoptosis of CD4+ T cells. Instead, it significantly suppressed the proliferation of conventional CD4+ T cells both in vitro and in vivo. Mechanistic study showed that DH-treated CD4+ T cells were partially arrested at the G2/M phase of the cell cycle because of the enhanced inhibitory phosphorylation of Cdc2 (Tyr15). In addition, we demonstrated that treatment with DH significantly ameliorated EAU in mice through suppressing the proliferation of autoreactive antigen specific CD4+ T cells. Taken together, the current study indicates that DH-mediated suppression of CD4+ T cell proliferation may provide a promising therapeutic strategy for treating CD4+ T cell-mediated diseases.

Unique microRNA alterations in hepatocellular carcinomas arising either spontaneously or due to chronic exposure to Ginkgo biloba extract (GBE) in B6C3F1/N mice.

Ginkgo biloba extract (GBE) is used in traditional Chinese medicine as a herbal supplement for improving memory. Exposure of B6C3F1/N mice to GBE in a 2-year National Toxicology Program (NTP) bioassay resulted in a dose-dependent increase in hepatocellular carcinomas (HCC). To identify key microRNAs that modulate GBE-induced hepatocarcinogenesis, we compared the global miRNA expression profiles in GBE-exposed HCC (GBE-HCC) and spontaneous HCC (SPNT-HCC) with age-matched vehicle control normal livers (CNTL) from B6C3F1/N mice. The number of differentially altered miRNAs in GBE-HCC and SPNT-HCC was 74 (52 up and 22 down) and 33 (15 up and 18 down), respectively. Among the uniquely differentially altered miRNAs in GBE-HCC, miR-31 and one of its predicted targets, Cdk1 were selected for functional validation. A potential miRNA response element (MRE) in the 3'-untranslated regions (3'-UTR) of Cdk1 mRNA was revealed by in silico analysis and confirmed by luciferase assays. In mouse hepatoma cell line HEPA-1 cells, we demonstrated an inverse correlation between miR-31 and CDK1 protein levels, but no change in Cdk1 mRNA levels, suggesting a post-transcriptional effect. Additionally, a set of miRNAs (miRs-411, 300, 127, 134, 409-3p, and 433-3p) that were altered in the GBE-HCCs were also altered in non-tumor liver samples from the 90-day GBE-exposed group compared to the vehicle control group, suggesting that some of these miRNAs could serve as potential biomarkers for GBE exposure or hepatocellular carcinogenesis. These data increase our understanding of miRNA-mediated epigenetic regulation of GBE-mediated hepatocellular carcinogenesis in B6C3F1/N mice.

Thermal cycling as a novel thermal therapy to synergistically enhance the anticancer effect of propolis on PANC­1 cells.

Hyperthermia (HT) has shown potential in cancer therapy. In particular, it appears to sensitize cancer cells to chemotherapy. However, a major concern associated with HT is that the thermal dosage applied to the tumor cells may also harm the normal tissue cells. Besides, the drugs used in HT are conventional chemotherapy drugs, which may cause serious side effects. The present study demonstrated a novel methodology in HT therapy called thermal cycle (TC)­HT. With this strategy, a therapeutic window with a maximum synergistic effect was created by combining TC­HT with natural compounds, with minimal unwanted cell damage. The natural compound propolis was selected, and the synergistic anticancer effect of TC­HT and propolis was investigated in pancreatic cancer cells. The present results demonstrated for the first time that TC­HT could enhance the anticancer effect of propolis on PANC­1 cancer cells through the mitochondria­dependent apoptosis pathway and cell cycle arrest. Combined treatment greatly suppressed mitochondrial membrane potential, which is an important indicator of damaged and dysfunctional mitochondria. Furthermore, the cell cycle­regulating protein cell division cycle protein 2 was downregulated upon combined treatment, which prevented cellular progression into mitosis. The present study offers the first report, to the best of our knowledge, on the combination of TC­HT with a natural compound for pancreatic cancer treatment. It is anticipated that this methodology may be a starting point for more sophisticated cancer treatments and may thereby improve the quality of life of many patients with cancer.

Supercritical CO2 fluid extraction of croton crassifolius Geisel root: Chemical composition and anti-proliferative, autophagic, apoptosis-inducing, and related molecular effects on A549 tumour cells.

BACKGROUND: The use of plant essential oils as pharmaceuticals is a fast-growing market especially in China. Throughout the 20th century, a rapid increase took place in the use of many essential oil-derived products in the medicinal industry as nutraceuticals, medicinal supplements, and pharmaceuticals. PURPOSE: The objective of this study was to explore the chemical composition of Croton crassifolius essential oil as well as its potential anti-tumour properties and related anti-proliferative, autophagic, and apoptosis-inducing effects. METHODS: Supercritical CO2 fluid extraction technology was used to extract CCEO and the chemical constituents of the essential oil were identified by comparing the retention indices and mass spectra data taken from the NIST library with those calculated based on the C7-C40 n-alkanes standard. The cytotoxic activity and anti-proliferative effects of CCEO were evaluated against five cancer cell lines and one normal human cell line via CCK-8 assays. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of CCEO treatments in controlling cancer cell proliferation was assessed by cell cycle analysis, clonal formation assays, RT-qPCR, and western blot analysis. Autophagic and apoptosis-inducing effects of oils and the associated molecular mechanisms were assessed by flow cytometry, cell staining, reactive oxygen species assays, RT-qPCR, and western blot analysis. CONCLUSION: Forty compounds representing 92.90% of the total oil were identified in CCEO. The results showed that CCEO exerted a measurable selectivity for cancer cell lines, especially for A549 with the lowest IC50 value of 25.00 ± 1.62 µg/mL. Assessment of the anti-proliferative effects of CCEO on A549 cells showed that the oil inhibited cell proliferation and colony formation in a dose- and time-dependent manner. Investigation of the molecular mechanisms of cell cycle regulation confirmed that the oil arrested A549 cells in G2/M phase by decreasing the expression of cyclin B1-CDK1 and cyclin A-CDK1 and increasing the expression of cyclin-dependent kinase inhibitor (CKI) P21 at both the transcriptional and translational levels. Autophagy staining assays and western blot analysis revealed that CCEO promoted the formation of autophagic vacuoles in A549 cells and increased the expression of autophagy-related proteins beclin-1 and LC3-II in a dose-dependent manner. A series of apoptosis analyses indicated that CCEO induces apoptosis through a mitochondria-mediated intrinsic pathway. This study revealed that CCEO is a promising candidate for development into an anti-tumour drug of the future.

Anti-proliferative effect of 8α-tigloyloxyhirsutinolide-13-O-acetate (8αTGH) isolated from Vernonia cinerea on oral squamous cell carcinoma through inhibition of STAT3 and STAT2 phosphorylation.

BACKGROUND: The high mortality rate of oral cancers has stimulated the search for effective herbal medicines and their pharmacological targets. Vernonia cinerea, a perennial tropical herb, is wildly used as a traditional folk medicine for treatment of intestinal diseases and various skin diseases in addition to possessing anti-cancer activity. However, the effect of 8α-tigloyloxyhirsutinolide-13-O-acetate (8αTGH) as a major sesquiterpene lactone compound found in V. cinerea and the underlying mechanism of its action on oral cancer cells remains unknown. PURPOSE: To investigate the anti-cancer activity of 8αTGH extracted from V. cinerea and the underlying mechanism of its action in oral cancer cells. METHODS: The anti-proliferative effect of 8αTGH on oral squamous cell carcinoma (HSC4) and lung carcinoma (A549) was determined using the SRB colorimetric method. The molecular mechanism of 8αTGH was explored using kinase inhibitors, followed by Western blotting or RT-qPCR. Flow cytometry and Western blotting were used to assess cell cycle arrest. RESULTS: 8αTGH inhibited cancer cell growth more effectively on HSC4 than A549 and was much less effective on tested normal oral cells. 8αTGH inhibited STAT3 phosphorylation on both cancer cells. Notably, 8αTGH was able to suppress the constantly activated STAT2 found only in HSC4. The STAT2 inhibition by 8αTGH consequently caused down-regulation of ISG15 and ISG15 conjugates. As a result, decreased expression of CDK1/2 and Cyclin B1 was detected leading to G2/M cell cycle arrest. CONCLUSION: 8αTGH isolated from V. cinerea preferentially inhibits the proliferation of oral cancer cells by causing G2/M cell cycle arrest via inhibition of both STAT3 and STAT2 phosphorylation. The results provide molecular bases for developing 8αTGH as a drug candidate or a complementary treatment of oral cancer.

Cyclin B1/CDK1-regulated mitochondrial bioenergetics in cell cycle progression and tumor resistance.

A mammalian cell houses two genomes located separately in the nucleus and mitochondria. During evolution, communications and adaptations between these two genomes occur extensively to achieve and sustain homeostasis for cellular functions and regeneration. Mitochondria provide the major cellular energy and contribute to gene regulation in the nucleus, whereas more than 98% of mitochondrial proteins are encoded by the nuclear genome. Such two-way signaling traffic presents an orchestrated dynamic between energy metabolism and consumption in cells. Recent reports have elucidated the way how mitochondrial bioenergetics synchronizes with the energy consumption for cell cycle progression mediated by cyclin B1/CDK1 as the communicator. This review is to recapitulate cyclin B1/CDK1 mediated mitochondrial activities in cell cycle progression and stress response as well as its potential link to reprogram energy metabolism in tumor adaptive resistance. Cyclin B1/CDK1-mediated mitochondrial bioenergetics is applied as an example to show how mitochondria could timely sense the cellular fuel demand and then coordinate ATP output. Such nucleus-mitochondria oscillation may play key roles in the flexible bioenergetics required for tumor cell survival and compromising the efficacy of anti-cancer therapy. Further deciphering the cyclin B1/CDK1-controlled mitochondrial metabolism may invent effect targets to treat resistant cancers.

Inhibition of Cytomegalovirus Replication with Extended-Half-Life Synthetic Ozonides.

Artesunate (AS), a semisynthetic artemisinin approved for malaria therapy, inhibits human cytomegalovirus (HCMV) replication in vitro, but therapeutic success in humans has been variable. We hypothesized that the short in vivo half-life of AS may contribute to the different treatment outcomes. We tested novel synthetic ozonides with longer half-lives against HCMV in vitro and mouse cytomegalovirus (MCMV) in vivo Screening of the activities of four ozonides against a pp28-luciferase-expressing HCMV Towne recombinant identified OZ418 to have the best selectivity; its effective concentration inhibiting viral growth by 50% (EC50) was 9.8 ± 0.2 µM, and cytotoxicity in noninfected human fibroblasts (the concentration inhibiting cell growth by 50% [CC50]) was 128.1 ± 8.0 µM. In plaque reduction assays, OZ418 inhibited HCMV TB40 in a concentration-dependent manner as well as a ganciclovir (GCV)-resistant HCMV isolate. The combination of OZ418 and GCV was synergistic in HCMV inhibition in vitro Virus inhibition by OZ418 occurred at an early stage and was dependent on the cell density at the time of infection. OZ418 treatment reversed HCMV-mediated cell cycle progression and correlated with the reduction of HCMV-induced expression of pRb, E2F1, and cyclin-dependent kinases 1, 2, 4, and 6. In an MCMV model, once-daily oral administration of OZ418 had significantly improved efficacy against MCMV compared to that of twice-daily oral AS. A parallel pharmacokinetic study with a single oral dose of OZ418 or AS showed a prolonged plasma half-life and higher unbound concentrations of OZ418 than unbound concentrations of AS. In summary, ozonides are proposed to be potential therapeutics, alone or in combination with GCV, for HCMV infection in humans.

Effect of methanol extract of Schisandrae Fructus on high fat diet induced hyperlipidemic mice.

OBJECTIVE: To investigate the effects and molecular targets of Schisandrae Fructus (SF) methanol extract (SFme) in mice with hyperlipidemia induced by high fat diet. METHODS: We observed changes in body weight, blood serum content of total cholesterol, high-density lipoprotein (HDL)-cholesterol, and triglyceride. The extent of accumulation of lipid peroxide due to lipid metabolism disorder also evaluated by measuring malondialdehyde (MDA) level. In addition, after getting gene expression in hepatic tissues, target protein of SFme was identified using a protein interaction database. RESULTS: SFme significantly decreased total cholesterol and triglyceride levels without alteration of body weight in mice, and the liver content of MDA was statistically decreased by SFme. And expression changes of cyclin- dependent kinase 1 (Cdk1) and leucine-rich repeat kinase 2 (Lrrk2) were restored by SFme. CONCLUSION: The effect of SFme on the high- fat-diet induced hyperlipidemia via decreasing total cholesterol and triglyceride levels may involve the expression of Cdk1 and Lrrk2 proteins.

YLT-11, a novel PLK4 inhibitor, inhibits human breast cancer growth via inducing maladjusted centriole duplication and mitotic defect.

Polo-like kinase 4 (PLK4) is indispensable for precise control of centriole duplication. Abnormal expression of PLK4 has been reported in many human cancers, and inhibition of PLK4 activity results in their mitotic arrest and apoptosis. Therefore, PLK4 may be a valid therapeutic target for antitumor therapy. However, clinically available small-molecule inhibitors targeting PLK4 are deficient and their underlying mechanisms still remain not fully clear. Herein, the effects of YLT-11 on breast cancer cells and the associated mechanism were investigated. In vitro, YLT-11 exhibited significant antiproliferation activities against breast cancer cells. Meanwhile, cells treated with YLT-11 exhibited effects consistent with PLK4 kinase inhibition, including dysregulated centriole duplication and mitotic defects, sequentially making tumor cells more vulnerable to chemotherapy. Furthermore, YLT-11 could strongly regulate downstream factors of PLK4, which was involved in cell cycle regulation, ultimately inducing apoptosis of breast cancer cell. In vivo, oral administration of YLT-11 significantly suppressed the tumor growth in human breast cancer xenograft models at doses that are well tolerated. In summary, the preclinical data show that YLT-11 could be a promising candidate drug for breast tumor therapy.

The ethanol extraction of prepared Psoralea corylifolia induces apoptosis and autophagy and alteres genes expression assayed by cDNA microarray in human prostate cancer PC-3 cells.

Prostate cancer is the most common male reproductive system cancer. The prevalence of prostate cancer in Europe and the United States is higher than that in the Asian region. However, the treatment of prostate cancer remains unsatisfactory. Psoralea corylifolia has been used to cure this disease as Chinese medicine in the Asian region. In this study, we analyzed the components of ethanol extraction of unprepared and prepared P. corylifolia by HPLC. Psoralen and isopsoralen content from the prepared P. corylifolia is twofold higher than that from unprepared, so we use the prepared extraction in this study. However, the effects of the ethanol extraction of P. corylifolia (PCE) on PC-3 human prostate cancer cells remain unclear. PC-3 cells were treated with PCE for different time periods and cells were examined for cell morphological change and total viable cells by using contrast phase microscopy and flow cytometer, respectively. Results indicated that PCE induced cell morphological changes and cytotoxic effect in PC-3 cells in dose-dependent manners. PCE induced chromatin condensation of PC-3 cells dose-dependently. PCE also induced apoptosis and autophagy in PC-3 by western blotting and acridine orange (AO) staining, respectively. Furthermore, a complementary DNA microarray analysis demonstrated that PCE treatment led to 944 genes upregulation and 872 genes downregulation. For example, the DNA damage-associated gene DNA-damage-inducible transcript 3 (DDIT 3) had a 62.1-fold upregulation and CDK1 2.68-fold downregulation. The differential genes were classified according to the Gene Ontology. Furthermore, GeneGo software was used for the key genes involved and their possible interaction pathways. Those genes were affected by P. corylifolia, which provided information for the understanding of the antiprostate cancer mechanism at the genetic level and provide additional targets for the treatments of human prostate cancer.

Sanyang Xuedai enhances the radiosensitivity of human non-small cell lung cancer cells via increasing iNOS/NO production.

OBJECTIVE: In this research, we aimed at finding out how San Yang Xue Dai (SYKT) promotes the radiosensitivity of non-small cell lung cancer (NSCLC) cell line NCI-H460. METHODS: Survival rate of NSCLC cells (A549, NCI-H460, NCI-H1650 and NCI-H1975) after the SYKT treatment or irradiation (IR) was calculated by the MTT assay. The radiosensitization of SYKT (0.5 g/mL and 1.0 g/mL) on cell line NCI-H460 and the radioresistant cell line NCI-H460R was studied by MTT assay and clone formation assay. The protein expression levels of iNOS, Cyclin B1 and CDC2 were determined by western blot, and the expression of NO was measured by Griess method. Finally, cell cycle and apoptotic rate of NSCLC cell line NCI-H460 were accessed by flow cytometry assay. BrdU staining was also applied to detect the cell proliferation after IR with or without SYKT treatment. RESULTS: The IC10 value of SYKT for NCI-H460 cells was 1.03 g/mL. After 1.0 g/mL SYKT treatment, the radiosensitivity of NCI-H460R cells was enhanced. The level of iNOS in the cells was found decreased after IR. We also found that SYKT could enhance iNOS and NO expressions while inhibit cyclin B1 and CDC2 expressions in radiation resistant cells. Combining ß-irradiation with SYKT caused cell cycle arrest in G2/M phase and increased cell apoptosis. CONCLUSION: SYKT resensitized radioresistant NCI-H460R cells via increasing cell apoptosis and cell cycle arrest. This was due to an elevated NO level caused by accumulating iNOS and effects of SYKT on radiosensitization of NSCLC should be further investigated in clinical application.

Effect of Huaier On the Proliferation of Mesangial Cells in Anti-Thy-1 Nephritis.

BACKGROUND/AIMS: To determine whether an aqueous extract of Trametes robiniophila Murr. (Huaier) suppresses anti-Thy-1 mesangial proliferative glomerulonephritis (MsPGN) in vivo and platelet-derived growth factor (PDGF)-BB-induced mesangial cell proliferation in vitro. METHODS: Male Wistar rats were randomly categorized into 5 groups: Sham, Thy-1, and 3 Huaier-treated groups (low, medium, and high dose). Two weeks after treatment, urinary proteins were quantified and renal pathological changes were examined. MAX interactor 1 (Mxi-1) and proliferating cell nuclear antigen (PCNA) expression levels in isolated glomeruli, rat mesangial cell viability, cell-cycle distribution, and cell-cycle pathways were assessed. RESULTS: Huaier diminished the proliferative damages and urinary protein secretion in Thy-1 rats. PCNA was downregulated, whereas Mxi-1 was upregulated in the isolated glomeruli of Huaier-treated groups compared with the Thy-1 group. Huaier inhibited PDGF-BB- stimulated proliferation of rat mesangial cells in a time- and dose-dependent manner (50% inhibitory concentration = 6.19 mg/mL) and induced G2 cell-cycle arrest. Cell-cycle pathway proteins were downregulated, whereas Mxi-1 was upregulated in Huaier-treated mesangial cells compared with PDGF-BB-stimulated cells. CONCLUSION: Huaier reduces urinary protein excretion and relieves hyperplasia in mesangial cells in anti-Thy-1 MsPGN as well as inhibits PDGF-BB-stimulated proliferation and DNA synthesis of rat mesangial cells in vitro, suggesting its novel therapeutic potential in MsPGN.

Prim-O-glucosylcimifugin induces cell cycle arrest and apoptosis in acute lymphoblastic leukemia cells.

Prim-O-glucosylcimifugin is a major constituent in Radix Saposhnikovia that has been long used for the treatment of pyrexia, rheumatism, and cancer in traditional Chinese medicine. However, the molecular and cellular mechanisms remain unknown regarding the therapeutic effect of prim-O-lucosylcimifugin. Here, we investigated the effects of prim-O-glucosylcimifugin on cell cycle progression and apoptosis in human acute lymphoblastic leukemia cells. Prim-O-glucosylcimifugin treatment resulted in marked increases in cell apoptosis and cell cycle arrest at the G2/M phase. Mechanistically, prim-O-glucosylcimifugin induced the degradation of ß-tubulin and downregulated phosphorylated CDK1 levels, a molecular indicator in the G2/M cell cycle arrest. Furthermore, activation of caspase-3, caspase-8, and caspase-9 was involved in the prim-O-glucosylcimifugin-induced apoptosis. Our study reveals the anticancer activity of prim-O-glucosylcimifugin and the potential underlying mechanisms.

Anthocyanins from roselle extract arrest cell cycle G2/M phase transition via ATM/Chk pathway in p53-deficient leukemia HL-60 cells.

Cell cycle regulation is an important issue in cancer therapy. Delphinidin and cyanidin are two major anthocyanins of the roselle plant (Hibiscus sabdariffa). In the present study, we investigated the effect of Hibiscus anthocyanins (HAs) on cell cycle arrest in human leukemia cell line HL-60 and the analyzed the underlying molecular mechanisms. HAs extracted from roselle calyces (purity 90%) markedly induced G2/M arrest evaluated with flow cytometry analysis. Western blot analyses revealed that HAs (0.1-0.7 mg mL-1 ) induced G2/M arrest via increasing Tyr15 phosphorylation of Cdc2, and inducing Cdk inhibitors p27 and p21. HAs also induced phosphorylation of upstream signals related to G2/M arrest such as phosphorylation of Cdc25C tyrosine phosphatase at Ser216, increasing the binding of pCdc25C with 14-3-3 protein. HAs-induced phosphorylation of Cdc25C could be activated by ATM checkpoint kinases, Chk1, and Chk2. We first time confirmed that ATM-Chk1/2-Cdc25C pathway as a critical mechanism for G2/M arrest in HAs-induced leukemia cell cycle arrest, indicating that this compound could be a promising anticancer candidate or chemopreventive agents for further investigation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1290-1304, 2017.

Hyperthermia enhances 17-DMAG efficacy in hepatocellular carcinoma cells with aggravated DNA damage and impaired G2/M transition.

Due to the lack of effective treatment, hepatocellular carcinoma (HCC) is one of the malignancies with low survival rates worldwide. Combination of hyperthermia and chemotherapy has shown promising results in several abdominal tumours, but high expression of HSP90 in tumours attenuated the efficacy of hyperthermia. Thus a combination of hyperthermia and inhibition of HSP90 might be a feasible therapeutic strategy for HCC. One hepatic cell line (L02) and two HCC cell lines (Huh7 and HepG2) were heated at 42 °C for 0, 0.5 or 4 h with or without 100 nM 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). HCC cells of the combination group exhibited more G2/M arrest and higher apoptotic rates which might result from suffering from more reactive oxygen species and serious DNA damage. Heat shock/17-DMAG co-treatment of HCC cells also destabilized CDK1, Cyclin B1 and CDC25C with a concomitant decreased proportion of cells in the M phase. Furthermore, co-treatment impaired the interaction of HSP90α with CDC37 and with CDK1, accompanied with decreased soluble CDK1. Combination of 17-DMAG with a 1.5-h whole body hyperthermia treatment attenuated tumour growth in xenograft mice models. These results suggest hyperthermia sensitize HCC to 17-DMAG, and combination of hyperthermia with 17-DMAG might be a potential therapeutic strategy for HCC.

Sulforaphane inhibits proliferation and invasive activity of everolimus-resistant kidney cancer cells in vitro.

Although the mechanistic target of rapamycin (mTOR) inhibitor, everolimus, has improved the outcome of patients with renal cell carcinoma (RCC), improvement is temporary due to the development of drug resistance. Since many patients encountering resistance turn to alternative/complementary treatment options, an investigation was initiated to evaluate whether the natural compound, sulforaphane (SFN), influences growth and invasive activity of everolimus-resistant (RCCres) compared to everolimus-sensitive (RCCpar) RCC cell lines in vitro. RCC cells were exposed to different concentrations of SFN and cell growth, cell proliferation, apoptosis, cell cycle, cell cycle regulating proteins, the mTOR-akt signaling axis, adhesion to human vascular endothelium and immobilized collagen, chemotactic activity, and influence on surface integrin receptor expression were investigated. SFN caused a significant reduction in both RCCres and RCCpar cell growth and proliferation, which correlated with an elevation in G2/M- and S-phase cells. SFN induced a marked decrease in the cell cycle activating proteins cdk1 and cyclin B and siRNA knock-down of cdk1 and cyclin B resulted in significantly diminished RCC cell growth. SFN also modulated adhesion and chemotaxis, which was associated with reduced expression of the integrin subtypes α5, α6, and ß4. Distinct differences were seen in RCCres adhesion and chemotaxis (diminished by SFN) and RCCpar adhesion (enhanced by SFN) and chemotaxis (not influenced by SFN). Functional blocking of integrin subtypes demonstrated divergent action on RCC binding and invasion, depending on RCC cell sensitivity to everolimus. Therefore, SFN administration could hold potential for treating RCC patients with established resistance towards everolimus.

KCa3.1 channel inhibition sensitizes malignant gliomas to temozolomide treatment.

Malignant gliomas are among the most frequent and aggressive cerebral tumors, characterized by high proliferative and invasive indexes. Standard therapy for patients, after surgery and radiotherapy, consists of temozolomide (TMZ), a methylating agent that blocks tumor cell proliferation. Currently, there are no therapies aimed at reducing tumor cell invasion. Ion channels are candidate molecular targets involved in glioma cell migration and infiltration into the brain parenchyma. In this paper we demonstrate that: i) blockade of the calcium-activated potassium channel KCa3.1 with TRAM-34 has co-adjuvant effects with TMZ, reducing GL261 glioma cell migration, invasion and colony forming activity, increasing apoptosis, and forcing cells to pass the G2/M cell cycle phase, likely through cdc2 de-phosphorylation; ii) KCa3.1 silencing potentiates the inhibitory effect of TMZ on glioma cell viability; iii) the combination of TMZ/TRAM-34 attenuates the toxic effects of glioma conditioned medium on neuronal cultures, through a microglia dependent mechanism since the effect is abolished by clodronate-induced microglia killing; iv) TMZ/TRAM-34 co-treatment increases the number of apoptotic tumor cells, and the mean survival time in a syngeneic mouse glioma model (C57BL6 mice implanted with GL261 cells); v) TMZ/TRAM-34 co-treatment reduces cell viability of GBM cells and cancer stem cells (CSC) freshly isolated from patients.Taken together, these data suggest a new therapeutic approach for malignant glioma, targeting both glioma cell proliferating and migration, and demonstrate that TMZ/TRAM-34 co-treatment affects both glioma cells and infiltrating microglia, resulting in an overall reduction of tumor cell progression.