A network pharmacology- and transcriptomics-based investigation reveals an inhibitory role of ß-sitosterol in glioma via the EGFR/MAPK signaling pathway.

Glioma is characterized by rapid cell proliferation, aggressive invasion, altered apoptosis and a poor prognosis. ß-Sitosterol, a kind of phytosterol, has been shown to possess anticancer activities. Our current study aims to investigate the effects of ß-sitosterol on gliomas and reveal the underlying mechanisms. Our results show that ß-sitosterol effectively inhibits the growth of U87 cells by inhibiting proliferation and inducing G2/M phase arrest and apoptosis. In addition, ß-sitosterol inhibits migration by downregulating markers of epithelial-mesenchymal transition (EMT). Mechanistically, network pharmacology and transcriptomics approaches illustrate that the EGFR/MAPK signaling pathway may be responsible for the inhibitory effect of ß-sitosterol on glioma. Afterward, the results show that ß-sitosterol effectively suppresses the EGFR/MAPK signaling pathway. Moreover, ß-sitosterol significantly inhibits tumor growth in a U87 xenograft nude mouse model. ß-Sitosterol inhibits U87 cell proliferation and migration and induces apoptosis and cell cycle arrest in U87 cells by blocking the EGFR/MAPK signaling pathway. These results suggest that ß-sitosterol may be a promising therapeutic agent for the treatment of glioma.

Hdac1-deficiency affects the cell cycle axis Cdc25-Cdk1 causing impaired G2/M phase progression and reduced cardiomyocyte proliferation in zebrafish.

Zebrafish have the ability to fully regenerate their hearts after injury since cardiomyocytes subsequently dedifferentiate, re-enter cell cycle, and proliferate to replace damaged myocardial tissue. Recent research identified the reactivation of dormant developmental pathways during cardiac regeneration in adult zebrafish, suggesting pro-proliferative pathways important for developmental heart growth to be also critical for regenerative heart growth after injury. Histone deacetylase 1 (Hdac1) was recently shown to control both, embryonic as well as adult regenerative cardiomyocyte proliferation in the zebrafish model. Nevertheless, regulatory pathways controlled by Hdac1 are not defined yet. By analyzing RNA-seq-derived transcriptional profiles of the Hdac1-deficient zebrafish mutant baldrian, we here identified DNA damage response (DDR) pathways activated in baldrian mutant embryos. Surprisingly, although the DDR signaling pathway was transcriptionally activated, we found the complete loss of protein expression of the known DDR effector and cell cycle inhibitor p21. Consequently, we observed an upregulation of the p21-downstream target Cdk2, implying elevated G1/S phase transition in Hdac1-deficient zebrafish hearts. Remarkably, Cdk1, another p21-but also Cdc25-downstream target was downregulated. Here, we found the significant downregulation of Cdc25 protein expression, explaining reduced Cdk1 levels and suggesting impaired G2/M phase progression in Hdac1-deficient zebrafish embryos. To finally prove defective cell cycle progression due to Hdac1 loss, we conducted Cytometer-based cell cycle analyses in HDAC1-deficient murine HL-1 cardiomyocytes and indeed found impaired G2/M phase transition resulting in defective cardiomyocyte proliferation. In conclusion, our results suggest a critical role of Hdac1 in maintaining both, regular G1/S and G2/M phase transition in cardiomyocytes by controlling the expression of essential cell cycle regulators such as p21 and Cdc25.

Inhibitory Effect of Salvia miltiorrhiza Extract and Its Active Components on Cervical Intraepithelial Neoplastic Cells.

The effective treatment of cervical intraepithelial neoplasia (CIN) can prevent cervical cancer. Salvia miltiorrhiza is a medicinal and health-promoting plant. To identify a potential treatment for CIN, the effect of S. miltiorrhiza extract and its active components on immortalized cervical epithelial cells was studied in vitro. The H8 cell was used as a CIN model. We found that S. miltiorrhiza extract effectively inhibited H8 cells through the CCK8 method. An HPLC-MS analysis revealed that S. miltiorrhiza extract contained salvianolic acid H, salvianolic acid A, salvianolic acid B, monomethyl lithospermate, 9‴-methyl lithospermate B, and 9‴-methyl lithospermate B/isomer. Salvianolic acid A had the best inhibitory effect on H8 cells with an IC50 value of 5.74 ± 0.63 µM. We also found that the combination of salvianolic acid A and oxysophoridine had a synergistic inhibitory effect on H8 cells at molar ratios of 4:1, 2:1, 1:1, 1:2, and 1:4, with salvianolic acid A/oxysophoridine = 1:2 having the best synergistic effect. Using Hoechst33342, flow cytometry, and Western blotting analysis, we found that the combination of salvianolic acid A and oxysophoridine can induce programmed apoptosis of H8 cells and block the cell cycle in the G2/M phase, which was correlated with decreased cyclinB1 and CDK1 protein levels. In conclusion, S. miltiorrhiza extract can inhibit the growth of H8 cells, and the combination of salvianolic acid A (its active component) and oxysophoridine has a synergistic inhibitory effect on H8 cells and may be a potential treatment for cervical intraepithelial neoplasia.

Anticancer Effects and Molecular Action of 7-α-Hydroxyfrullanolide in G2/M-Phase Arrest and Apoptosis in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is a breast cancer subtype characterized by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 expression. TNBC cells respond poorly to targeted chemotherapies currently in use and the mortality rate of TNBC remains high. Therefore, it is necessary to identify new chemotherapeutic agents for TNBC. In this study, the anti-cancer effects of 7-α-hydroxyfrullanolide (7HF), derived from Grangea maderaspatana, on MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cells were assessed using MTT assay. The mode of action of 7HF in TNBC cells treated with 6, 12 and 24 µM of 7HF was determined by flow cytometry and propidium iodide (PI) staining for cell cycle analysis and annexin V/fluorescein isothiocyanate + PI staining for detecting apoptosis. The molecular mechanism of action of 7HF in TNBC cells was investigated by evaluating protein expression using proteomic techniques and western blotting. Subsequently, 7HF exhibited the strongest anti-TNBC activity toward MDA-MB-468 cells and a concomitantly weak toxicity toward normal breast cells. The molecular mechanism of action of low-dose 7HF in TNBC cells primarily involved G2/M-phase arrest through upregulation of the expression of Bub3, cyclin B1, phosphorylated Cdk1 (Tyr 15) and p53-independent p21. Contrastingly, the upregulation of PP2A-A subunit expression may have modulated the suppression of various cell survival proteins such as p-Akt (Ser 473), FoxO3a and ß-catenin. The concurrent apoptotic effect of 7HF on the treated cells was mediated via both intrinsic and extrinsic modes through the upregulation of Bax and active cleaved caspase-7-9 expression and downregulation of Bcl-2 and full-length caspase-7-9 expression. Notably, the proteomic approach revealed the upregulation of the expression of pivotal protein clusters associated with G1/S-phase arrest, G2/M-phase transition and apoptosis. Thus, 7HF exhibits promising anti-TNBC activity and at a low dose, it modulates signal transduction associated with G2/M-phase arrest and apoptosis.

Non-alkylator anti-glioblastoma agents induced cell cycle G2/M arrest and apoptosis: Design, in silico physicochemical and SAR studies of 2-aminoquinoline-3-carboxamides.

Malignant gliomas are the most common brain tumors, with generally dismal prognosis, early clinical deterioration and high mortality. Recently, 2-aminoquinoline scaffold derivatives have shown pronounced activity in central nervous system disorders. We herein reported a series of 2-aminoquinoline-3-carboxamides as novel non-alkylator anti-glioblastoma agents. The synthesized compounds showed comparable activity to cisplatin against glioblastoma cell line U87 MG in vitro. Among them, we found that 6a displayed good inhibitory activity against A172 and U118 MG glioblastoma cell lines and induced cell cycle arrest in the G2/M phase and apoptosis in U87 MG by flow cytometry analysis. Additionally, 6a displayed low cytotoxicity to several normal human cell lines. In silico study showed 6a had promising physicochemical properties and was predicted to cross the blood-brain barrier. Moreover, preliminary structure-activity relationships are also investigated, shedding light on further modifications towards more potent agents on this series of compounds. Our results suggest this compound has a promising potential as an anti-glioblastoma agent with a differential effect between tumor and non-malignant cells.

Anticancer effect of sodium metavanadate on murine breast cancer both in vitro and in vivo.

Sodium metavanadate (NaVO3) exhibits important physiological effects including insulin-like, chemoprevention and anticancer activity. However, the effects of NaVO3 on breast cancer and underlying mechanisms are still unclear. In this study, our results revealed that NaVO3 was able to inhibit proliferation of murine breast cancer cells 4T1 with IC50 value of 8.19 µM and 1.92 µM at 24 h and 48 h, respectively. The mechanisms underlying the inhibition activity were that NaVO3 could increase reactive oxygen species (ROS) level in a concentration-dependent way, arrest cells at G2/M phase, diminish the mitochondrial membrane potential (MMP), finally promote the progress of apoptosis. Furthermore, NaVO3 also exhibited a dose-dependent anticancer activity in breast cancer-bearing mice that led to the shrinkage of tumor volume (about 50%), lower microvessel density, less propagating cells and more apoptotic cells in vivo, as compared to the saline group. Therefore, NaVO3 may act as a potential chemotherapeutic agent in breast cancer treatment.

Double-component diazeniumdiolate derivatives as anti-cancer agents.

In this study, we synthesized a series of double-component O2-aryl diazeniumdiolate (DDNO) derivatives, of which each molecule can release up to four nitric oxide molecules. These compounds showed cytotoxic activities to cancer cells, such as human leukemia, breast cancer and lung cancer. Among them, compound 1 (DDNO-1) showed the highest specific activity to human leukemia cells. It induced cell apopotosis and arrest cell cycle of G2/M phase. The JNK and p38 protein kinases were activated by compound 1 to induce cancer cell apoptosis. Compound 1 also increased pro-apoptotic Bax level, which is a same function compared to a reported NO donor, JS-K. More interestingly, it decreased the level of an anti-apoptotic member Bcl-2, which is an opposite effect compared to JS-K. Compound 1 could be developed as a new anti-cancer agent since it increases the Bax/Bcl-2 ratio to overcome the drug resistance.

LW-213, a newly synthesized flavonoid, induces G2/M phase arrest and apoptosis in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell neoplasm characterized by an uncontrolled proliferation of moderately and well differentiated cells of the granulocytic lineage. LW-213, a newly synthesized flavonoid compound, was found to exert antitumor effects against breast cancer through inducing G2/M phase arrest. We investigated whether LW-213 exerted anti-CML effects and the underlying mechanisms. We showed that LW-213 inhibited the growth of human CML cell lines K562 and imatinid-resistant K562 (K562r) in dose- and time-dependent manners with IC50 values at the low µmol/L levels. LW-213 (5, 10, 15 µM) caused G2/M phase arrest of K562 and K562r cells via reducing the activity of G2/M phase transition-related proteins Cyclin B1/CDC2 complex. LW-213 treatment induced apoptosis of K562 and K562r cells via inhibiting the expression of CDK9 through lysosome degradation, thus leading to the suppression of RNAPII phosphorylation, down-regulation of a short-lived anti-apoptic protein MCL-1. The lysosome inhibitor, NH4Cl, could reverse the anti-CML effects of LW-213 including CDK9 degradation and apoptosis. LW-213 treatment also degraded the downstream proteins of BCR-ABL1, such as oncoproteins AKT, STAT3/5 in CML cells, which was blocked by NH4Cl. In primary CML cells and CD34+ stem cells, LW-213 maintained its pro-apoptotic activity. In a K562 cells-bearing mice model, administration of LW-213 (2.5, 5.0 mg/kg, ip, every other day for 4 weeks) dose-dependently prolonged the survival duration, and significantly suppressed huCD45+ cell infiltration and expression of MCL-1 in spleens. Taken together, our results demonstrate that LW-213 may be an efficient agent for CML treatment.

MicroRNA-301b-3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4.

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR-301b-3p functions as a driver in various types of human cancer. However, the expression pattern of miR-301b-3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR-301b-3p expression was significantly up-regulated in HCC tissues compared to adjacent non-tumour tissues. Clinical association analysis revealed that the high level of miR-301b-3p closely correlated with large tumour size and advanced tumour-node-metastasis stages. Importantly, the high miR-301b-3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR-301b-3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR-301b-3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR-301b-3p directly bond to 3'UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down-regulated and inversely correlated with miR-301b-3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR-301b-3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR-301b-3p is highly expressed in HCC. miR-301b-3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.

CtIP promotes G2/M arrest in etoposide-treated HCT116 cells in a p53-independent manner.

Acquired resistance to cytotoxic antineoplastic agents is a major clinical challenge in tumor therapy; however, the mechanisms involved are still poorly understood. In this study, we show that knockdown of CtIP, a corepressor of CtBP, promotes cell proliferation and alleviates G2/M phase arrest in etoposide (Eto)-treated HCT116 cells. Although the expression of p21 and growth arrest and DNA damage inducible α (GADD45a), which are important targets of p53, was downregulated in CtIP-deficient HCT116 cells, p53 deletion did not affect G2/M arrest after Eto treatment. In addition, the phosphorylation levels of Ser317 and Ser345 in Chk1 and of Ser216 in CDC25C were lower in CtIP-deficient HCT116 cells than in control cells after Eto treatment. Our results indicate that CtIP may enhance cell sensitivity to Eto by promoting G2/M phase arrest, mainly through the ATR-Chk1-CDC25C pathway rather than the p53-p21/GADD45a pathway. The expression of CtIP may be a useful biomarker for predicting the drug sensitivity of colorectal cancer cells.

MiR-219-5p suppresses cell proliferation and cell cycle progression in esophageal squamous cell carcinoma by targeting CCNA2.

BACKGROUND: We investigated the potential regulatory role of miR-219-5p in esophageal squamous cell carcinoma (ESCC) and looked at the underlying mechanisms in ESCC. METHODS: Real-time PCR was used to determine the levels of miR-219-5p in ESCC tissues and cell lines. The effects of miR-219-5p and cyclin A2 (CCNA2) on cell proliferation and cell cycle progression were evaluated using MTT, colony formation and flow cytometry assays with ESCC cell lines EC9706 and TE-9. Bioinformatics techniques and the luciferase reporter assay were applied to validate CCNA2 as the miR-219-5p target in ESCC cells. The mRNA and protein levels of CCNA2 were measured using real-time PCR and western blotting. RESULTS: MiR-219-5p expression was significantly lower in ESCC tissues and cells than in healthy tissues. Upregulation of miR-219-5p repressed cell proliferation and induced cell cycle arrest at the G2/M phase. CCNA2 was identified and confirmed as a direct downstream target of miR-219-5p and its expression negatively correlated with miR-219-5p profiles in ESCC tissues. Knockdown of CCNA2 potentiated the effects of miR-219-5p on cell proliferation and cell cycle distribution. CONCLUSIONS: Our results demonstrate that miR-219-5p might function as a tumor suppressor by directly targeting CCNA2 expression. It could serve as a new therapeutic target for ESCC.

Sodium Fluoride Arrests Renal G2/M Phase Cell-Cycle Progression by Activating ATM-Chk2-P53/Cdc25C Signaling Pathway in Mice.

BACKGROUND/AIMS: Excessive fluoride intake can induce cytotoxicity, DNA damage and cell-cycle changes in many tissues and organs, including the kidney. However, the underlying molecular mechanisms of fluoride-induced renal cell-cycle changes are not well understood at present. In this study, we used a mouse model to investigate how sodium fluoride (NaF) induces cell-cycle changes in renal cells. METHODS: Two hundred forty ICR mice were randomly assigned to four equal groups for intragastric administration of NaF (0, 12, 24 and 48 mg/kg body weight/day) for 42 days. Kidneys were taken to measure changes of the cell-cycle at 21 and 42 days of the experiment, using flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot methods. RESULTS: NaF, at more than 12 mg/kg body weight, induced G2/M phase cell-cycle arrest in the renal cells, which was supported by the finding of significantly increased percentages of renal cells in the G2/M phase. We found also that G2/M phase cell-cycle arrest was accompanied by up-regulation of p-ATM, p-Chk2, p-p53, p-Cdc25C, p-CDK1, p21, and Gadd45a protein expression levels; up-regulation of ATM, Chk2, p53, p21, and Gadd45a mRNA expression levels; down-regulation of CyclinB1, mdm2, PCNA protein expression levels; and down-regulation of CyclinB1, CDK1, Cdc25C, mdm2, and PCNA mRNA expression levels. CONCLUSION: In this mouse model, NaF, at more than 12 mg/ kg, induced G2/M phase cell-cycle arrest by activating the ATM-Chk2-p53/Cdc25C signaling pathway, which inhibits the proliferation of renal cells and development of the kidney. Activation of the ATM-Chk2-p53/Cdc25C signaling pathway is the mechanism of NaF-induced renal G2/M phase cell-cycle arrest in this model.

Adenovirus armed with VGLL4 selectively kills hepatocellular carcinoma with G2/M phase arrest and apoptosis promotion.

The Vestigial-Like Family Member 4 (VGLL4) functions as a native inhibitor of cell proliferation and tumor growth through multiple signaling pathways. We first discovered that VGLL4 causes G2/M phase arrest in hepatocellular carcinoma (HCC) cells. Then, we designed a novel survivin-regulated oncolytic adenovirus Ad-sp-VGLL4 carrying the VGLL4 gene. Ad-sp-VGLL4 exerted high HCC-targeting-selectivity but is less harmful to normal cells. This adenovirus construction enhanced antitumor activity due to G2/M phase arrest and enhanced apoptosis. It's also indicated that Ad-sp-VGLL4 could suppress the growth of transplanted tumor of HCC in vivo experiment. Taken together, our results suggest that Ad-sp-VGLL4 possesses strong antitumor capacity and has great potential use for HCC therapy.

Histone deacetylase 7 silencing induces apoptosis and autophagy in salivary mucoepidermoid carcinoma cells.

BACKGROUND: The overexpression of histone deacetylases (HDACs) has been observed in many cancers, and inhibition of specific HDACs has emerged as a new target for cancer therapy. We found that HDAC7 expression was selectively reduced by HDAC inhibitor apicidin in salivary mucoepidermoid carcinoma (MEC) cells. Here, we show that HDAC7 suppression has a potent antitumor effect in MEC cells. METHODS: Histone deacetylases7 was knocked down using HDAC7 siRNAs, and cell proliferation was quantified. Cell cycle progression, apoptosis, and autophagy were measured by flow cytometry and immunoblotting. RESULTS: Histone deacetylases 7 siRNAs inhibited cell proliferation and c-Myc expression, increased p27 expression, and caused G2/M phase cell cycle arrest in both YD-15 and Mc3 cells. HDAC7 silencing increased the sub-G1 population, Annexin V positive apoptotic cells and cleaved caspase3 levels. HDAC7 silencing induced an increase in autophagic markers, number of acidic vesicular organelles, and LC3B II levels, and decrease in p62 levels. HDAC7 siRNAs reduced the activation of ERK. HDAC7 knockdown resulted in growth inhibition through G2/M phase cell cycle arrest and induced both apoptosis and autophagy in MEC cells. CONCLUSIONS: This study indicates that inhibition of HDAC7 might become a novel and effective therapeutic approach for treating to MEC.

Ziyuglycoside I Inhibits the Proliferation of MDA-MB-231 Breast Carcinoma Cells through Inducing p53-Mediated G2/M Cell Cycle Arrest and Intrinsic/Extrinsic Apoptosis.

BACKGROUND: Due to the aggressive clinical behavior, poor outcome, and lack of effective specific targeted therapies, triple-negative breast cancer (TNBC) has currently been recognized as one of the most malignant types of tumors. In the present study, we investigated the cytotoxic effect of ziyuglycoside I, one of the major components extracted from Chinese anti-tumor herbal Radix Sanguisorbae, on the TNBC cell line MDA-MB-231. METHODS: The underlying molecular mechanism of the cytotoxic effect ziyuglycoside I on MDA-MB-231 cells was investigated with cell viability assay, flow cytometric analysis and Western blot. RESULTS: Compared to normal mammary gland Hs 578Bst cells, treatment of ziyuglycoside I resulted in a significant growth inhibitory effect on MDA-MB-231 cells. Ziyuglycoside I induced the G2/M phase arrest and apoptosis of MDA-MB-231 cells in a dose-dependent manner. These effects were found to be partially mediated through the up-regulation of p53 and p21WAF1, elevated Bax/Bcl-2 ratio, and the activation of both intrinsic (mitochondrial-initiated) and extrinsic (Fas/FasL-initiated) apoptotic pathways. Furthermore, the p53 specific siRNA attenuated these effects. CONCLUSION: Our study suggested that ziyuglycoside I-triggered MDA-MB-231 cell cycle arrest and apoptosis were probably mediated by p53. This suggests that ziyuglycoside I might be a potential drug candidate for treating TNBC.

Evaluation of the cytotoxicity, cell-cycle arrest, and apoptotic induction by Euphorbia hirta in MCF-7 breast cancer cells.

CONTEXT: Euphorbia hirta L. (Euphorbiaceae) has been used as a folk remedy in Southeast Asia for the treatment of various ailments. OBJECTIVE: The current study evaluates the cytotoxicity, cell-cycle arrest, and apoptotic induction by E. hirta in MCF-7 breast cancer cells. MATERIALS AND METHODS: Cytotoxic activity of methanol extract of whole part of E. hirta was determined by the MTT assay at various concentrations ranging from 1.96 to 250.00 µg/mL in MCF-7 cells. Cell morphology was assessed by light and fluorescence microscopy. Apoptosis and cell-cycle distribution were determined by annexin V staining and flow cytometry. DNA fragmentation, caspase activity, and reactive oxygen species (ROS) assays were performed using the commercially available kits. To identify the cytotoxic fraction, E. hirta extract was subjected to bioassay-guided fractionation. RESULTS: Euphorbia hirta exhibited significant inhibition of the survival of MCF-7 cells and the half inhibitory concentration (IC50) values was 25.26 µg/mL at 24 h. Microscopic studies showed that E. hirta-treated cells exhibited marked morphological features characteristic of apoptosis. Euphorbia hirta extract also had an ignorable influence on the LDH leakage and generating intracellular ROS. The flow cytometry study confirmed that E. hirta extract induced apoptosis in MCF-7 cells. Euphorbia hirta also resulted in DNA fragmentation in MCF-7 cells. Moreover, E. hirta treatment resulted in the accumulation of cells at the S and G2/M phases as well as apoptosis. The caspase activity study revealed that E. hirta extract induced apoptosis through the caspase-3-independent pathway by the activation of caspase-2, 6, 8, and 9. Euphorbia hirta hexane fraction, namely HFsub4 fraction, demonstrated highest activity among all the fractions tested with an IC50 value of 10.01 µg/mL at 24 h. DISCUSSION AND CONCLUSION: This study revealed that E. hirta induced apoptotic cell death and suggests that E. hirta could be used as an apoptosis-inducing anticancer agent for breast cancer treatment with further detailed studies.

Induction of Mitochondrial Dependent Apoptosis in Human Leukemia K562 Cells by Meconopsis integrifolia: A Species from Traditional Tibetan Medicine.

OBJECTIVES: Meconopsis integrifolia (M. integrifolia) is one of the most popular members in Traditional Tibetan Medicine. This study aimed to investigate the anticancer effect of M. integrifolia and to detect the underlying mechanisms of these effects. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and trypan blue assay were used to evaluate the cytotoxicity of M. integrifolia. Changes in cell nuclear morphology and reactive oxygen species (ROS) level were observed by fluorescent microscopy. Apoptosis ratio, DNA damage and mitochondrial membrane potential (MMP) loss were analyzed by flow cytometry. Western blotting assay was adopted to detect the proteins related to apoptosis. Immunofluorescence was used to observe the release of cytochrome C. RESULTS: The obtained data revealed that M. integrifolia could significantly inhibit K562 cell viability, mainly by targeting apoptosis induction and cell cycle arrest in G2/M phase. Collapse in cell morphology, chromatin condensation, DNA damage and ROS accumulation were observed. Further mechanism detection revealed that mitochondrion might be a key factor in M. integrifolia-induced apoptosis. CONCLUSIONS: M. integrifolia could induce mitochondria mediated apoptosis and cell cycle arrest in G2/M phase with little damage to normal cells, suggesting that M. integrifolia might be a potential and efficient anticancer agent that deserves further investigation.

Telocinobufagin, a PLK1 suppressor that inhibits tumor growth and metastasis by modulating CDC25c and CTCF in HNSCC cells.

BACKGROUND: The high metastasis and mortality rates of head and neck squamous cell carcinoma (HNSCC) urgently require new treatment targets and drugs. A steroidal component of ChanSu, telocinobufagin (TBG), was verified to have anti-cancer effects in various tumors, but its activity and mechanism in anti-HNSCC were still unknown. PURPOSE: This study tried to demonstrate the anti-tumor effect of TBG on HNSCC and verify its potential mechanism. METHODS: The effect of TBG on cell proliferation and metastasis were performed and the TBG changed genes were detected by RNA-seq analysis in HNSCC cells. The GSEA and PPI analysis were used to identify the pathways targeted for TBG-regulated genes. Meanwhile, the mechanism of TBG on anti-proliferative and anti-metastasis were investigated in vitro and in vivo. RESULTS: The in vitro and in vivo experiments confirmed that TBG has favorable anti-tumor effects by induced G2/M phase arrest and suppressed metastasis in HNSCC cells. Further RNA-seq analysis demonstrated the genes regulated by TBG were enriched at the G2/M checkpoint and PLK1 signaling pathway. Then, the bioinformatic analysis of clinical data found that high expressed PLK1 were closely associated with poor overall survival in HNSCC patients. Furthermore, PLK1 directly and indirectly modulated G2/M phase and metastasis (by regulated CTCF) in HNSCC cells, simultaneously. TBG significantly inhibited the protein levels of PLK1 in both phosphorylated and non-phosphorylated forms and then, in one way, inactivated PLK1 failed to activate G2/M phase-related proteins (including CDK1, CDC25c, and cyclin B1). In another way, be inhibited PLK1 unable promote the nuclear translocation of CTCF and thus suppressed HNSC cell metastasis. In contrast, the anti-proliferative and anti-metastasis effects of TBG on HNSCC cell were vanished when cells high-expressed PLK1. CONCLUSION: The present study verified that PLK1 mediated TBG induced anti-tumor effect by modulated G2/M phase and metastasis in HNSCC cells.

PLK1 inhibition leads to mitotic arrest and triggers apoptosis in cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is a lethal cancer originating from the epithelial cells within the bile duct and ranks as the second most prevalent form of liver cancer in Thailand. Polo-like kinase 1 (PLK1), a protein serine/threonine kinase, regulates a number of steps in cell mitosis and is upregulated in several types of cancer, including CCA. Our previous study identified PLK1 as a biomarker of the C1 subtype, correlating with poor prognosis in intrahepatic CCA. The present study aimed to examine the effect of PLK1 inhibition on CCA cells. Different CCA cell lines developed from Thai patients, HuCCA1, KKU055, KKU100 and KKU213A, were treated with two PLK1 inhibitors, BI2536 and BI6727, and were transfected with small interfering RNA, followed by analysis of cell proliferation, cell cycle distribution and cell apoptosis. It was discovered that BI2536 and BI6727 inhibited cell proliferation and caused G2/M-phase arrest in CCA cells. Furthermore, the number of total apoptotic cells was increased in PLK1 inhibitor-treated CCA cells. The expression levels of mitotic proteins, aurora kinase A, phosphorylated PLK1 (T210) and cyclin B1, were augmented in PLK1-inhibited CCA cells. Additionally, inhibition of PLK1 led to increased DNA damage, as determined by the upregulated levels of γH2AX and increased cleavage of poly (ADP-ribose) polymerase, an apoptotic marker. These results suggested that inhibiting PLK1 prolonged mitotic arrest and subsequently triggered cell apoptosis. Validation of the antiproliferative effects of PLK1 inhibition was accomplished through silencing of the PLK1 gene. In conclusion, targeting PLK1 provided promising results for further study as a potential candidate for targeted therapy in CCA.

Libertellenone T, a Novel Compound Isolated from Endolichenic Fungus, Induces G2/M Phase Arrest, Apoptosis, and Autophagy by Activating the ROS/JNK Pathway in Colorectal Cancer Cells.

Colorectal cancer (CRC) is the third most deadly type of cancer in the world and continuous investigations are required to discover novel therapeutics for CRC. Induction of apoptosis is one of the promising strategies to inhibit cancers. Here, we have identified a novel compound, Libertellenone T (B), isolated from crude extracts of the endolichenic fungus from Pseudoplectania sp. (EL000327) and investigated the mechanism of action. CRC cells treated by B were subjected to apoptosis detection assays, immunofluorescence imaging, and molecular analyses such as immunoblotting and QRT-PCR. Our findings revealed that B induced CRC cell death via multiple mechanisms including G2/M phase arrest caused by microtubule stabilization and caspase-dependent apoptosis. Further studies revealed that B induced the generation of reactive oxygen species (ROS) attributed to activating the JNK signaling pathway by which apoptosis and autophagy was induced in Caco2 cells. Moreover, B exhibited good synergistic effects when combined with the well-known anticancer drug, 5-FU, and another cytotoxic novel compound D, which was isolated from the same crude extract of EL000327. Overall, Libertellenone T induces G2/M phase arrest, apoptosis, and autophagy via activating the ROS/JNK pathway in CRC. Thus, B may be a potential anticancer therapeutic against CRC that is suitable for clinical applications.