TUBA1A licenses APC/C-mediated mitotic progression to drive glioblastoma growth by inhibiting PLK3.

Glioblastoma (GBM) is the most common, aggressive, and chemorefractory primary brain tumor in adults. Identifying novel drug targets is crucial for GBM treatment. Here, we demonstrate that tubulin alpha 1a (TUBA1A) is significantly upregulated in GBM compared to low-grade gliomas (LGG) and normal tissues. High TUBA1A expression is associated with poor survival in GBM patients. TUBA1A knockdown results in mitotic arrest and reduces tumor growth in mice. TUBA1A interacts with the polo-like kinase 3 (PLK3) in the cytoplasm to inhibit its activation. This interaction licenses activation of the anaphase-promoting complex or cyclosome (APC/C) to ensure proper Foxm1-mediated metaphase-to-anaphase transition and mitotic exit. Overall, our findings demonstrate that targeting TUBA1A attenuates GBM cell growth by suppressing mitotic progression in a PLK3-dependent manner.

Selective elimination of CML stem/progenitor cells by picropodophyllin in vitro and in vivo is associated with p53 activation.

Chronic myeloid leukemia (CML) is a hematologic malignancy originating from BCR-ABL oncogene-transformed hematopoietic stem cells (HSCs) known as leukemia stem cells (LSCs). Therefore, targeting LSCs is of primary importance to eradicate CML. The present study demonstrates that picropodophyllin (PPP) effectively induces apoptosis and inhibits colony formation in CML stem/progenitor cells as well as quiescent CML progenitors resistant to imatinib therapy, while sparing normal hematopoietic cells in vitro. Administration of PPP in vivo markedly diminishes CML stem/progenitor cells in a transgenic mouse model of CML by inhibition of cell proliferation and enhancement of apoptosis in LSK cells, and significantly improves survival of CML mice. Furthermore, PPP treatment preferentially leads to transcriptional activation of p53 in CML but not normal CD34+ cells, upregulation of p53 protein in LSCs-enriched Sca-1+ cells from CML mice, and increased phosphorylation of p53 and upregulation of Bax protein in Ku812 cells. These results suggest that the inhibitory effects of PPP on CML stem/progenitor cells are associated with selective activation of p53 pathway and propose that PPP is a potent agent that selectively targets CML LSCs, and may be of value in the CML therapy.

[Effect of Etoposide on Elimination of Chronic Myeloid Leukemia Stem Cells by Imatinib in Vivo].

OBJECTIVE: To investigate the effect of etoposide (ETO) on elimination of chronic myeloid leukemia (CML) stem cells by imatinib mesylate(IM) in vivo. METHODS: SCL-tTA/BCR-ABL mice were used as CML animal model. Flow cytometry was used to assess the effect of ETO alone or in combination with IM on the number of leukemia stem cell （LSC） in bone marrow and spleen, and peripheral blood neutrophils in CML mice and normal control FVB mice. RESULTS: The results showed that in CML mice, the number and proportion of LSC in bone marrow and the proportion of neutrophils in peripheral blood decreased significantly after ETO and IM combined treatment, and the degree of decrease was more significant than that of both alone. While in wild type FVB mice, the combination of ETO and IM showed no significant effect on the number and proportion of LSK cells in bone marrow and the proportion of neutrophils in spleen. CONCLUSION: ETO can selectively enhance elimination of CML LSC by IM in vivo.

Selective and effective targeting of chronic myeloid leukemia stem cells by topoisomerase II inhibitor etoposide in combination with imatinib mesylate in vitro.

Imatinib mesylate (IM) and other BCR-ABL tyrosine kinase inhibitors (TKIs) have improved chronic myeloid leukemia (CML) patient survival markedly but fail to eradicate quiescent CML leukemia stem cells (LSCs). Thus, strategies targeting LSCs are required to induce long-term remission and achieve cure. Here, we investigated the ability of topoisomerase II (Top II) inhibitor etoposide (Eto) to target CML LSCs. Treatment with Eto combined with IM markedly induced apoptosis in primitive CML CD34+ CD38- stem cells resistant to eradication by IM alone, but not in normal hematopoietic stem cells, CML and normal mature CD34- cells, and other leukemia and lymphoma cell lines. The interaction of IM and Eto significantly inhibited phosphorylation of PDK1, AKT, GSK3, S6, and ERK proteins; increased the expression of pro-apoptotic gene Bax; and decreased the expression of anti-apoptotic gene c-Myc in CML CD34+ cells. Top II inhibitors treatment represents an attractive approach for targeting LSCs in CML patients undergoing TKIs monotherapy.

Silencing of miR-21 sensitizes CML CD34+ stem/progenitor cells to imatinib-induced apoptosis by blocking PI3K/AKT pathway.

BCR-ABL tyrosine kinase inhibitor imatinib fails to eradicate leukemia stem cells (LSCs), the underlying mechanisms maintaining CML LSCs remain poorly understood. Here, we showed that transient inhibition of miR-21 by antagomiR-21 markedly increased imatinib-induced apoptosis in CML, but not normal CD34+ stem/progenitor cells. Furthermore, PI3K inhibitors also significantly sensitized CML CD34+ cells to imatinib-induced apoptosis. MiR-21 or PI3K inhibitor in combination with imatinib treatment significantly decreased AKT phosphorylation and c-Myc expression than either agent did alone, but did not affect Bim and Bcl-6 expresssion. These findings indicate that miR-21 is required for maintaining the imatinib-resistant phenotype of CML CD34+ cells through PI3K/AKT signaling pathway, thus providing the basis for a promising therapeutic approach to eliminate CML LSCs.

Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival.

The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control ß1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

Targeting miR-21 sensitizes Ph+ ALL Sup-b15 cells to imatinib-induced apoptosis through upregulation of PTEN.

Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cells are insensitive to BCR-ABL tyrosine kinase inhibitor imatinib, the underlying mechanisms remain largely unknown. Here, we showed that imatinib treatment induced significant upregulation of miR-21 and downregulation of PTEN in Ph+ ALL cell line Sup-b15. Transient inhibition of miR-21 resulted in increased apoptosis, PTEN upregulation and AKT dephosphorylation, whereas ectopic overexpression of miR-21 further conferred imatinib resistance. Furthermore, knockdown of PTEN protected the cells from imatinib-induced apoptosis achieved by inhibition of miR-21. Additionally, PI3K inhibitors also notably enhanced the effects of imatinib on Sup-b15 cells and primary Ph+ ALL cells similar to miR-21 inhibitor. Therefore, miR-21 contributes to imatinib resistance in Ph+ ALL cells and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to imatinib therapy.

[Gleevec induces apoptosis in K562 cells through activating caspase-3].

The present study is to elucidate the mechanisms underlying Gleevec-induced apoptosis of chronic myeloid leukemia (CML) K562 cells in vitro. The apoptotic cell death and cell cycle distribution after Gleevec treatment and the effect of PDCD4 siRNA on Gleevec-induced apoptosis of K562 cells were analyzed by flow cytometry. The effect of Gleevec on p-Crkl, caspase-3, PARP and PDCD4 protein levels, and the knockdown efficacy of PDCD4 siRNA were detected by Western blotting. The results showed that Gleevec dramatically suppressed the phosphorylation level of Crkl in a dose-dependent manner and induced significant apoptosis and G0/G1 cell cycle arrest of K562 cells in time- and dose-dependent manners. In addition, Gleevec activated caspase-3 and its downstream substrates PARP, and the caspase pan inhibitor Z-VAD-FMK (50 micromol x L(-1)) markedly reduced Gleevec-induced apoptosis from 47.97% +/- 10.56% to 31.05% +/- 9.206% (P < 0.05). Moreover, Gleevec significantly increased the protein expression of programmed cell death 4 (PDCD4). PDCD4 knockdown by siRNA reduced Gleevec-induced apoptosis from 46.97% +/- 14.32% to 42.8% +/- 11.43%. In summary, Gleevec induced apoptosis in K562 cells via caspase-3 activation.

Wedelia chinensis inhibits nasopharyngeal carcinoma CNE-1 cell growth by inducing G2/M arrest in a Chk1-dependent pathway.

Although Wedelia chinensis, an herb in traditional Chinese medicine, has been widely used for the treatment of inflammation, the effects of W. chinensis on cancer cell growth and the related molecular mechanisms behind these effects have largely remained unexplored to date. In the present study, W. chinensis plant extracts were obtained using either ethanol (E), petroleum ether (PE), ethyl acetate (EA) or butyl alcohol (BA). Then, extracts were examined for bioactivity in vitro via MTT assay in five human cancer cell lines. Our results showed that one subfraction of the EA extract (EA6) was cytotoxic to nasopharyngeal carcinoma (NPC) CNE-1 cells, among all cell lines evaluated. Treatment of CNE-1 cells with EA6 resulted in significant G2/M cell cycle arrest and modest apoptosis. EA6 induced Chk1 activation and inhibition of Chk1 in CNE-1 cells by RNA interference (RNAi) markedly abrogated EA6-mediated G2/M arrest and abolished EA6-induced cytotoxicity. EA6 treatment resulted in notable reduction of c-myc expression in CNE-1 cells, whereas silencing Chk1 inhibited such effects of EA6. Our results indicate that Chk1 is a novel molecular target of EA6 in NPC cells and also suggest an intervention strategy for NPC by EA6 exploring its molecular mechanisms of action.

Curcumin induces FasL-related apoptosis through p38 activation in human hepatocellular carcinoma Huh7 cells.

AIM: The aim of this study is to explore the underlying molecular mechanism of curcumin-induced apoptosis in human hepatocellular carcinoma (HCC) Huh7 cells. MAIN METHODS: Fas and FasL mRNA expression was analyzed by reverse transcription PCR. Western blot was applied to detect the protein expression of Bcl-2 family members, MAPK family members, c-Jun, c-Fos, ATF-2, caspase-3, PARP, TNF receptor family members and the respective ligands. Apoptotic cells were assayed with annexin V/PI double staining and flow cytometry. KEY FINDINGS: Curcumin treatment resulted in a fast and significant increase of Fas and Fas ligand (FasL) along with activation of caspase-3 and cleavage of PARP in Huh7 cells. Inhibition of caspase-3 activity by the specific inhibitor Z-DEVD-FMK rescued Huh7 cells from curcumin-induced apoptosis. Neutralization of FasL significantly protected the cells from curcumin-induced caspase-3 activation and apoptosis in a dose-dependent manner. Moreover, p38 was rapidly activated in response to curcumin, and inactivation of p38 by pharmacologic inhibitor SB203580 dramatically suppressed curcumin-induced FasL expression and apoptosis. SIGNIFICANCE: Our results demonstrated that curcumin induces apoptosis through p38-denpendent up-regulation of FasL in Huh7 cells.

A highly selective colorimetric chemodosimeter for fast and quantitative detection of hydrogen sulfide.

Based on the fact that hydrogen sulfide (H(2)S) possesses the smallest steric hindrance among thiols and the SH(-) group adds faster to an electron-poor C=C double bond, we designed and synthesized a tricyanoethylene-derived colorimetric chemodosimeter 1 for the fast and highly selective assay of H(2)S. Chemodosimeter 1 exhibited excellent water-solubility due to the introduction of two hydrophilic hydroxyl groups. Upon the addition of Na(2)S, chemodosimeter 1 showed a fast (complete within 400 s) and robust decrease of the absorption intensity (>97%), accompanied by a color change from red to colorless. Additionally, a linear relationship between absorption intensity and the added Na(2)S concentrations (0-130 µM) was observed in aqueous buffer solution (pH 7.4, 20 mM PBS). Importantly, the proposed paradigm in this paper, adoption of the tricyanoethylene derivative as a recognition receptor to distinguish H(2)S from other thiols and analytes, provides a promising methodology for the design of colorimetric and fluorescent chemodosimeters for fast determination of H(2)S.

Cell cycle-dependent subcellular distribution of ClC-3 in HeLa cells.

Chloride channel-3 (ClC-3) is suggested to be a component and/or a regulator of the volume-activated Cl(-) channel in the plasma membrane. However, ClC-3 is predominantly located inside cells and the role of intracellular ClC-3 in tumor growth is unknown. In this study, we found that the subcellular distribution of endogenous ClC-3 varied in a cell cycle-dependent manner in HeLa cells. During interphase, ClC-3 was distributed throughout the cell and it accumulated at various positions in different stages. In early G1, ClC-3 was mainly located in the nucleus. In middle G1, ClC-3 gathered around the nuclear periphery as a ring. In late G1, ClC-3 moved back into the nucleus, where it remained throughout S phase. In G2, ClC-3 was concentrated in the cytoplasm. When cells progressed from G2 to the prophase of mitosis, ClC-3 from the cytoplasm translocated into the nucleus. During metaphase and anaphase, ClC-3 was distributed throughout the cell except for around the chromosomes and was aggregated at the spindle poles and in between two chromosomes, respectively. ClC-3 was then again concentrated in the nucleus upon the progression from telophase to cytokinesis. These results reveal a cell cycle-dependent change of the subcellular distribution of ClC-3 and strongly suggest that ClC-3 has nucleocytoplasmic shuttling dynamics that may play key regulatory roles during different stages of the cell cycle in tumor cells.

Imidazo [4,5f][1,10] phenanthroline derivatives as inhibitor of c-myc gene expression in A549 cells via NF-κB pathway.

1,10-Phenanthroline has been shown to exhibit anticancer activity. Here, a series of imidazo [4,5f][1,10] phenanthroline derivatives 1-10 were synthesized and their biological activities were further elucidated. We found that 2-(4-Brominephenyl)-imidazo [4,5f][1,10] phenanthroline (compound 3) possessed potent antiproliferation activities again a variety of tumor cell lines using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Flow cytometric analysis revealed that compound 3 induced both through apoptosis and necrosis in human lung adenocarcinoma cell line, A549. Moreover, compound 3 treatment led to up-regulation of IκBα and down-regulation of p65 and c-myc in A549 cells. Taken together, these results suggested that compound 3 inhibited cell proliferation by suppression of NF-κB activity and down-regulation of c-myc gene expression and may be a candidate for further evaluation as a chemopreventive and chemotherapeutic agent for human cancers, especially for lung cancer.

Lack of association between stretch-activated and volume-activated Cl⁻ currents in hepatocellular carcinoma cells.

Stretch-activated chloride currents (I(Cl,SA) ) have been considered to be a component of volume-activated chloride currents (I(Cl,vol) ) for some time. This is due to a similarity in biophysical and pharmacological properties that involve a membrane curvature-induced mechanism and rearrangement of the cytoskeleton induced by cell swelling or membrane stretch. In the present study, we demonstrated that current density, along with the time taken from the activation of currents to the peak, were significantly different between the two currents, in highly metastatic human hepatocellular carcinoma cells. In addition, the activation of I(Cl,vol) or I(Cl,SA), induced maximally by hypotonic solutions or membrane stretch, respectively, did not affect the following activation of the other one. Moreover, neither inhibition of I(Cl,vol) by sh-ClC-3 transfection, nor functional blocking of I(Cl,vol) by intracellular dialysis of anti-ClC-3 antibody had an effect on the activation and properties of I(Cl,SA). Collectively, our results suggest that I(Cl,SA) is different from I(Cl,vol) in activation mechanism and/or in molecular entity responsible for formation of the currents. ClC-3 is involved in the activation of I(Cl,vol), but not of I(Cl,SA).

ClC-3 chloride channels are essential for cell proliferation and cell cycle progression in nasopharyngeal carcinoma cells.

ClC-3, a gene encoding a candidate protein for volume-activated chloride (C(-)) channels, may be involved in tumor development. Herein we report a study using an antisense "knock-down" strategy to investigate the mechanism by which ClC-3 affects cell proliferation in nasopharyngeal carcinoma CNE-2Z cells. With immunoblots and MTT assays we demonstrated that the expression of ClC-3 was cell cycle dependent and in a similar concentration-dependent manner, an antisense oligonucleotide specific for ClC-3 inhibited ClC-3 protein expression and cell proliferation. The expression level of ClC-3 correlated with cell proliferation. Moreover, in the cells exposed to a ClC-3 antisense oligonucleotide, the cloning efficiency was inhibited, and cells were arrested in the S phase. The ClC-3 antisense oligonucleotide inhibited the volume-activated C(-) current (I(Cl,vol)) and the regulatory volume decrease (RVD) in a concentration-dependent manner. Additionally, the I(Cl,vol) or RVD was positively correlated with cell proliferation in the treated cells. In conclusion, ClC-3 is involved in cell proliferation and cell cycle progression through a mechanism involving modulation of I(Cl,vol) and RVD. CIC-3 may represent a therapeutic target in human cancer.

[Anticancer activities of curcumin on human hepatocarcinoma cell line Sk-hep-1].

To study the anticancer activities of curcumin on human hepatocarcinoma cell line Sk-hep-1 and its related molecular mechanism which has not been elucidated. In the present study,we showed that curcumin inhibited proliferation of Sk-hep-1 cells in a dose-dependent manner through MTF assay. The effect of curcumin on apoptosis in Sk-hep-1 cells was investigated by DAPI staining and the various apoptosis was observed in hepatocarcinoma cell lines Sk-hep-1, HepG2 and Hep3B, but not in normal liver cell line Chang's liver with curcumin treatment. Cell cycle analysis results showed that curcumin treatment resulted in dramatic accumulation of Sk-hep-1 cells at the G0/G1 or G2/M phase. The effect of curcumin on the expression of anti-apoptosis genes (Survivin and BCl-xL) and drug resistance genes (DRG2 and MDR1) was studied by reverse transcription-polymerase chain reaction (RT-PCR). The expression of MDR1 mRNA was significantly decreased in Sk-hep-1 cells treated with curcumin, while no alterations in the amount of DRG2 and anti-apoptosis genes' mRNA levels were found. These results indicate that curcumin is able to inhibit proliferation and induce apoptosis in Sk-hep-1 cells and it may cause by down-regulating the expression of MDR1 mRNA.

Study on the anti-wear performance of Ni-base composite coating sucker joint that contains nano-diamond and nano-polytetrafluoroethylene.

With the development of oilfields, the problem of eccentric wear between casing and sucker rod in rod-pumped wells operation is more and more severe. Investigations on the eccentric wear show that the abrasion of sucker rod joint is more serious than the sucker rod itself. A new method of producing the Ni-base composite coating that contains nano-diamond and nano-polytetrafluoroethylene (PTFE) on sucker joint obtained by electrodeposition is presented in this paper. The test results show that the anti-wear performance and hardness of the sucker rod improve significantly with the increase of nano-diamond. The addition of nano-PTFE particle is useful in reducing the friction factor. Field tests demonstrate that the life of the sucker rod joint is increased and the maintenance cycle of the rod-pumped well is prolonged.

[Effect of p21-targeted shRNA on curcumin-induced apoptosis of human hepatoma Huh7 cells].

In the present study, shRNA plasmid of pSi-p21 targeting p21 mRNA was constructed and the effect of p21 shRNA on curcumin-induced apoptosis of human hepatoma Huh7 cells was investigated. The effect of curcumin on the expression of p21 mRNA and protein and the silence efficiency of pSi-p21 were detected with RT-PCR and Western blotting. The effect of pSi-p21 on curcumin-induced apoptosis of Huh7 cells was evaluated with DAPI staining. The results showed that curcumin significantly upregulated p21 mRNA and protein expression, which was knocked down by pSi-p21 of Huh7 cells. DAPI staining results showed that pSi-p21 significantly decreased curcumin-induced apoptosis of Huh7 cells. The data suggested that curcumin induced apoptosis of Huh7 cells via upregulation of p21 expression.

Volume-activated chloride channels contribute to cell-cycle-dependent regulation of HeLa cell migration.

The activation of volume-activated chloride Cl(-) channels has been implicated to play important roles in modulating cell cycle and cell migration. The aim of this study was to determine whether volume-activated Cl(-) channels are involved in cell-cycle-dependent regulation of cell migration in HeLa cells. Using techniques including cell-cycle synchronization, transwell migration assays and the patch-clamp technique, we demonstrate in this study that both the expression of volume-activated chloride current (I(Cl,vol)) and the potential of cell migration are cell-cycle-dependent; specifically, these events were high in G(0)/G(1) phase, low in S phase, and medium in G(2)/M phase. Moreover, the mean density of I(Cl,vol) was positively correlated to the rate of cell migration during cell-cycle progression. Additionally, endogenous suppression of I(Cl,vol) by transfecting cells with ClC-3 antisense oligonucleotides arrested cells in S phase and slowed cell migration. Collectively, our results suggest that volume-activated Cl(-) channels contribute to the cell-cycle-dependent regulation of cell migration.

[Effect of curcumin on JAK-STAT signaling pathway in hepatoma cell lines].

The effect of curcumin on JAK-STAT signaling pathway was investigated in hepatoma cell lines Huh7 and Hep3B. Curcumin inhibited cell proliferation and induced apoptosis of both cell lines, but Huh7 cells were more sensitive to curcumin than Hep3B cells. Curcumin (50 micromol x L(-1)) significantly increased phosphorylations of p38 (T180/Y182) and STAT-1 (S727) in Huh7 and Hep3B cells, and caused relocalization of phosphorylated-STAT-1 (Y701) from cytoplasm to nucleus in Hep3B cells. In addition, curcumin (25 and 50 micromol x L(-1)) dramatically suppressed the phosphorylation level of STAT-1 (Y701) and resulted in a significant reduction of nuclear phosphorylated-STAT-1 (Y701) in Huh7 cells.