Induction of apoptosis by capsaicin in A172 human glioblastoma cells

Capsaicin induced apoptosis of A172 human glioblastoma cells in a time- and dose-dependent manner. Neither capsazepine, a vanilloid receptor antagonist, nor bis-(o-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM), an intracellular Ca(2+) chelator, significantly inhibited the capsaicin-induced apoptosis, although capsaicin increased intracellular Ca(2+) level. Capsaicin markedly reduced the basal generation of reactive oxygen species (ROS) and lipid peroxidation. Exogenous application of H(2)O(2) significantly prevented the cells from the apoptosis by capsaicin. Treatment with N-acetyl cysteine alone induced both reduction of the basal production of ROS and apoptosis. Taken together, these results suggest that capsaicin induced apoptosis in A172 cells and that vanilloid receptors and intracellular Ca(2+) may not be involved in the apoptotic mechanism of capsaicin. Reduction of the basal generation of ROS may play a role in the induction of apoptosis by capsaicin.     

Apoptosis induced by hyperthermia in human glioblastoma cell line and murine glioblastoma

Apoptosis is a type of cell death that plays an important role in the early development and growth of normal tissue. It is modulated by a variety of stimuli. In multicellular organisms, homeostasis is maintained through a balance between cell proliferation and cell death. Recent evidence suggests that derangement of apoptosis also contributes to a number of human diseases including tumors.[1] More attention has been paid to cell proliferation rather than apoptosis in the study of tumorigenesis…     

Continuous internalization of TNF receptors in a human myosarcoma cell line

The cell dynamics of the receptor on tumor necrosis factor (TNF) were studied with the use of TNF-sensitive KYM cells derived from human myosarcoma. With receptor synthesis inhibited by cycloheximide, the half-life of the surface TNF receptor was 2h in the absence of TNF and 30min in its presence, suggesting that the TNF receptor was non-recycling and that its internalization was accelerated by TNF. During cell incubation with suppression of TNF receptor degradation by chloroquine, the number of surface TNF receptors remained approximately constants, but the total number of surface and internal TNF receptors increased gradually, at 3h reaching 1.5 times of the initial number, thus suggesting continuous synthesis, externalization, internalization, and degradation of the TNF receptor in the absence of cycloheximide. When the cells were incubated with 125I-TNF, the intracellular quantity of the pulse-labeled TNF-receptor complex promptly increased, reaching a maximum at 20 min, and then declining gradually. Thus, it was confirmed that the TNF receptor is internalized as a TNF-receptor complex in the presence of TNF. In incubation with suppression of protein synthesis by cycloheximide following surface TNF receptor digestion by trypsin, TNF receptors reappeared on the cell surface, increasing in the number to a peak level at 60 min and gradually decreasing. The cells previously exposed to cycloheximide with or without TNF showed no recurrence of surface TNF receptors, suggesting that the TNF receptor is non-recycling. The results thus suggest that the TNF receptor is continuously internalized and degraded intracellularly by lysosomes without being recycled regardless of the presence or absence of TNF, and further that its internalization is accelerated when it is part of the TNF-receptor complex.     

Phenoxazine derivatives induce caspase-independent cell death in human glioblastoma cell lines, A-172 and U-251 MG

The apoptotic effects of 2-amino-4,4alpha-dihydro-4alpha, 7-dimethyl-3H-phenoxazine-3-one (Phx-1) and 2-aminophenoxazine-3-one (Phx-3) on human glioblastoma cell lines, A-172 and U-251 MG were studied. These phenoxazines extensively decreased the viability of A-172 and U-251 MG cells (IC50 of Phx-1: 60 microM, in both lines; IC50 of Phx-3: 10 and 3 microM, for A-172 and U-251 cells, respectively). Phx-1 and Phx-3 increased the population of annexin V and PI double-positive cells in A-172 and U-251 MG cells, resulting in cell death at late stage apoptosis/necrosis. The activities of caspase-3/7 were greatly increased in A-172 and U-251 MG cells treated with Phx-1 or Phx-3. However, a pan-caspase inhibitor, z-VAD-fmk, failed to reverse the antiproliferative and apoptotic effects of Phx-1 and Phx-3 in both cell lines. In conclusion, Phx-1 and Phx-3 exert significant anti-cancer effects against human glioblastoma cell lines, A-172 and U-251 MG, mediated by the caspase-independent apoptotic cell death pathway.     

A new matrix metalloproteinase inhibitor SI-27 induces apoptosis in several human myeloid leukemia cell lines and enhances sensitivity to TNF alpha-induced apoptosis.

MMP inhibitors are used clinically for the stabilization of tumor growth, thus prolonging survival in cancer patients. However, their role in the treatment of hematopoietic malignancies remains unclear. In the present study, we investigated the effects of a new MMP inhibitor, SI-27, in hematopoietic malignancies. SI-27 alone induces apoptosis in several human myeloid leukemia cell lines such as U937, NB4, and HL60 cells by activating caspase 8, 9, and 3. Apoptosis was measured with annexin V positive staining, a drop in mitochondrial transmembrane potential (deltapsim), presence of hypodiploid DNA, and cleavage of PARP and IkappaBalpha. Furthermore, at lowered concentrations, which did not directly induce apoptosis, SI-27 acted to sensitize U937 cells and other cells to tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. The accumulation of membrane Fas, the Fas ligand, and TNFR1 were not apparent due to exposure to SI-27, and antagonistic anti-Fas or anti-Fas ligand antibodies did not block SI-27-induced apoptosis. Thus, SI-27-induced apoptosis is not mediated by the Fas pathway. These results suggest that MMP inhibitors, alone or in combination with other cytotoxic agents, can provide a unique method for treating acute myeloid leukemia, refractory to classical anti-cancer drugs, and may thus suppress recurrence.     

MiR-198 enhances temozolomide sensitivity in glioblastoma by targeting MGMT

Many studies have implicated operative length as a predictor of post-operative complications, including venous thromboembolism [deep vein thrombosis (DVT) and pulmonary embolism (PE)]. We analyzed the American College of Surgeons (ACS) National Surgical Quality Improvement Program (NSQIP) database from 2006 to 2014, to evaluate whether length of operation had a statistically significant effect on post-operative complications in patients undergoing surgical resection of meningioma. Patients were included for this study if they had a post-operative diagnosis of meningioma. Patient demographics, pre-operative comorbidities, and post-operative 30-day complications were analyzed. Of 3743 patients undergoing craniotomy for meningioma, 13.6?% experienced any complication. The most common complications and their median time to occurrence were urinary tract infection (2.6?%) at 10 days postoperatively (IQR 7–15), unplanned intubation (2.5?%) at 3 days (IQR 1–7), failure to wean from ventilator (2.4?%) at 2.0 days (IQR 2–4), and DVT (2.4?%) at 6 days (IQR 11–19). Postoperatively, 3.6?% developed VTE; 2.4?% developed DVT and 1.7?% developed PE. Multivariable analysis identified older age (third and upper quartile), obesity, preoperative ventilator dependence, preoperative steroid use, anemia, and longer operative time as significant risk factors for VTE. Separate multivariable logistic regression models demonstrated longer operative time as a significant risk factor for VTE, all complications, major complications, and minor complications. Meningioma resection is associated with various post-operative complications that increase patient morbidity and mortality risk. this large, multi-institutional patient sample, longer operative length was associated with increased risk for postoperative venous thromboembolisms, as well as major and minor complications.     

CARMA3 is overexpressed in human glioma and promotes cell invasion through MMP9 regulation in A172 cell line

Caspase recruitment domain-containing membrane-associated guanylate kinase protein 10 or CARMA3 (CARD10) is a recently characterized oncoprotein involved in the progression of several human malignancies. The present study aims to investigate the expression pattern and biological roles of CARMA3 protein in human glioma. CARMA3 expression was analyzed in 97 glioma specimens using immunohistochemistry. We observed negative staining in normal astrocytes and positive staining of CARMA3 in 25 out of 97 (25.8 %) glioma samples. Overexpression of CARMA3 correlated with tumor grade (p?<?0.001). Small interfering RNA knockdown was performed in A172 cell line with relatively high CARMA3 expression. Using colony formation assay and Matrigel invasion assay, we showed that CARMA3 depletion in A172 cell line inhibited cell proliferation and cell invasion. In addition, mRNA and protein levels of matrix metallopeptidase 9 (MMP9) were downregulated, indicating CARMA3 might regulate invasion through MMP9. In conclusion, CARMA3 serves as an oncoprotein in human glioma by regulating cell invasion, possibly through MMP9 regulation.     

Detection of a hypersialylated beta1 integrin endogenously expressed in the human astrocytoma cell line A172

Gliomas are the most common deadly brain tumors. Human cerebral tumors express high level of alpha5beta1 integrins. As a potential new target, alpha5beta1 was investigated here in two human astrocytoma cell lines, A172 and U87MG. We found that a hypersialylated beta1 integrin was endogenously expressed in A172 cells. It forms heterodimers with alpha5 subunits, localizes at the cell membrane and allows adhesion to fibronectin. This form of beta1 integrin was only recognized by the 9EG7 anti-beta1 antibody and appeared devoid of other specific antibody epitopes (12G10, TS2/16 and mAb13 shown here to be N-glycosylation sensitive). Overexpression of the beta1 integrin subunit in A172 cells not only increased the hypersialylated form but also led to the appearance of a non-hypersialylated beta1 form also addressed to the cell surface. Compared to wild-type A172 cells, beta1-A172 cells showed increased adhesion to fibronectin and decreased sensitivity to SJ749, a non-peptidic alpha5beta1 antagonist. In addition, beta1-A172 cells exhibited increased matrix dependence for normal cell cycling. Collectively, the data add new evidence for the role of beta1 glycosylation/sialylation in the regulation of integrin functions.     

Interferon-gamma enhances radiation-induced cell death via downregulation of Chk1

Interferon-gamma (IFNγ) is a cytokine with roles in immune responses as well as in tumor control. Interferon is often used in cancer treatment together with other therapies. Here we report a novel approach to enhancement of cancer cell killing by combined treatment of IFNγ with ionizing radiation. We found that IFNγ treatment alone in HeLa cells induced phosphorylation of Chk1 in a time- and dose-dependent manner, and resulted in cell arrest. Moreover IFNγ treatment was correlated with attenuation of Chk1 as the treatment shortened protein half-life of Chk1. As Chk1 is an essential cell cycle regulator for viability after DNA damage, attenuation of Chk1 by IFNγ pre-treatment in HeLa cells resulted in increased cell death following ionizing radiation about 2-folds than ionizing radiation treatment alone whereas IFNγ treatment alone had little effect on cell death. X-linked inhibitor of apoptosis-associated factor 1 (XAF1), an IFN-induced gene, seems to partly regulate IFNγ-induced Chk1 destabilization and radiation sensitivity because transient depletion of XAF1 by siRNA prevented IFNγ-induced Chk1 attenuation and partly protected cells from IFNγ-enhanced radiation cell killing. Therefore the results provide a novel rationale to combine IFNγ pretreatment and DNA-damaging anti-cancer drugs such as ionizing radiation to enhance cancer cell killing.     

Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines

Summary Smac/DIABLO is a mitochondrial protein released into cytosol during the progression of apoptosis. Smac/DIABLO promotes apoptosis by neutralizing the inhibitory effect of the inhibitor of apoptosis proteins (IAPs) on the processing and activity of the effecter of caspase. Here, we generated synthetic Smac peptide which possesses an IAP-binding domain and Drosophila antennapaedia penetration sequence, and examined whether it enhances the effect of the chemotherapeutic agent etoposide in the human glioblastoma cell line. Cellular uptake of Smac peptide in several glioma cell lines was most prominent at 6–12 h after addition. Caspase activity assay showed that our peptide successfully increased the activity of caspase-3 and caspase-9 in etoposide-induced apoptosis. In addition, Smac peptide increased the amount of cleaved PARP (poly ADP-ribose polymerase), but control peptides did not. Moreover, the addition of z-VAD-fmk, a caspase inhibitor, counterbalanced the effect of Smac peptide. Finally, we demonstrated that Smac peptide could enhance the growth inhibition effect of etoposide compared with control peptides. These results suggest that synthetic Smac peptide may be a new molecular targeting anti-tumor therapy for human glioblastoma.     

IGF-1R抑制剂联用可增强人非小细胞肺癌PC9/G细胞对吉非替尼的敏感性

目的:观察单用表皮生长因子受体(epidermal growth factor receptor, EGFR)的酪氨酸激酶抑制剂吉非替尼 (ge? tinib) 或联合胰岛素生长因子-1受体(insulin-like growth factor-1 receptor,IGF-1R)的酪氨酸激酶抑制剂AG1024作用于人非小细胞肺癌耐药株PC9/G细胞后,该细胞对吉非替尼耐药性的影响,并探讨IGF-1R与肿瘤细胞耐药的相关机制。方法:用吉非替尼和AG1024单独或联合作用于PC9/G细胞后,采用MTT法分别检测各组细胞的增殖情况,并利用中效原理判断两药联用的效果;FCM法检测各组细胞的凋亡情况;Western印迹法检测各组细胞的磷酸化EGFR(phosphorylated EGFR, p-EGFR)、磷酸化Akt(phosphorylated Akt,p-Akt)和磷酸化细胞外信号调节激酶(phosphorylated extracellular signal-regulated kinase,p-ERK)的表达水平。结果:吉非替尼和AG1024单独作用于PC9/G细胞后,均出现不同程度的细胞增殖抑制作用和细胞凋亡促进作用;而吉非替尼和AG1024联合作用,能更显著地抑制细胞增殖,且凋亡细胞显著增加(P<0.05)。 Western印迹法检测发现,联合用药组的p-EGFR、p-Akt和p-ERK蛋白表达量明显减少。结论:IGF-1R抑制剂AG1024和EGFR抑制剂吉非替尼联用具有较好的协同作用,可能通过抑制细胞增殖和促进细胞凋亡,提高耐药细胞对吉非替尼的敏感性。     

Selective beta2-adrenoceptor agonist enhances sensitivity to cisplatin in non-small cell lung cancer cell line

Cisplatin is a key drug in chemotherapy for lung cancer. It has been reported that intracellular accumulation of cisplatin is an important step as a determinant for resistance to cisplatin, which may be modulated by Na+, K+-ATPase activity. And it has been reported that isoproterenol, a beta-adrenoceptor agonist, enhances sensitivity to cisplatin in non-small cell lung cancer (NSCLC) cell lines. In this study, the effects of the selective beta1, beta2, and beta3-adrenoceptor agonists on membrane Na+, K+-ATPase activity and sensitivity to cisplatin were evaluated using human non-small cell lung cancer cell line. In the NSCLC cell line, sensitivity to cisplatin was improved by treatment with procaterol, a selective beta2-adrenoceptor agonist. Na+, K+-ATPase was activated and intracellular accumulation of cisplatin increased with the treatment. However, beta1 or beta3-adrenoceptor agonist did not modulate sensitivity to cisplatin or Na+, K+-ATPase activity. These results suggest that beta2-adrenoceptor may be one of the determinants for sensitivity to cisplatin in NSCLC. Exogenous beta2-adrenoceptor agonists may improve the antitumor effect of chemotherapy involving cisplatin.     

Halofuginone enhances the radiation sensitivity of human tumor cell lines

Transforming growth factor beta (TGF-β) is implicated in radiation-induced fibrosis of normal tissues in patients receiving radiotherapy. Inhibiting the TGF-β signaling pathway by various means has been shown to reduce radiation-induced fibrosis in pre-clinical studies. The present study evaluated the effects of interfering with the TGF-β signaling pathway on the radiosensitivity of selected human tumor cell lines using the plant-derived alkaloid, halofuginone. Halofuginone treatment inhibited cell growth, halted cell cycle progression, decreased radiation-induced DNA damage repair, and decreased TGF-β receptor II protein levels, leading to increased cellular radiosensitization. These data further support the goal of manipulating the TGF-β pathway to achieve a positive increase in the therapeutic gain in clinical radiotherapy.     

Effect of hyperthermia on cyclin B expression in a human glioblastoma cell line

Summary Effects of hyperthermia on the cell kinetics of glioblastoma cells were investigated using flow cytometry. Pulse-labeling with 5-bromodeoxyuridine (BUdR) and chasing of the labeled cells revealed temporary accumulation of the labeled cells in G₂M phase and a reduction of DNA synthesis.The level of cyclin B rises rapidly in G₂ phase and falls at the end of mitosis in normal cycling cells. Cyclin B binds to p34^cdc2, resulting in histone kinase activity which is necessary for the initiation of mitosis. The amount of p34^cdc2 remains constant throughout the cell cycle. The level of cyclin B was measured using an anti-cyclin B antibody and flow cytometry in order to investigate the cause of the G₂ accumulation induced by hyperthermia. A low level of cyclin B, in comparison with that of normal cycling cells, persisted for more than 3 h after hyperthermia. These results indicate that the temporary accumulation of cells in G₂M phase after hyperthermia may be caused, at least in part, by an insufficient level of cyclin B.     

Recombinant human stem cell factor stimulates growth of a human glioblastoma cell line expressing c-kit protooncogene.

The growth of a panel of eight different human glioblastoma cell lines was examined in a human tumor cloning assay in agar, a tritiated thymidine uptake assay, and by counting cell numbers, in cultures performed in the absence or presence of increasing concentrations (1 to 100 ng/ml) of recombinant human stem cell factor (SCF). Growth of 7 of 8 cell lines was not significantly and reproducibly affected by recombinant human SCF. However, growth of the CRL 1620 cell line could be stimulated up to 5-fold by the cytokine. In contrast to the other cell lines investigated, CRL 1620 expressed the c-kit protooncogene assessed on the mRNA and protein level. Furthermore, SCF-induced proliferation of CRL 1620 cells was sensitive to the tyrosine kinase inhibitor erbstatin. Our data suggest that SCF can be operative in growth modulation of malignant cells outside the hematopoietic system, and this finding should be further studied for its possible clinical implications.     

Contribution of autophagic cell death to p53-dependent cell death in human glioblastoma cell line SF126

Apoptosis and autophagic cell death are programmed cell deaths that are involved in cell survival, growth, development and carcinogenesis. p53, the most extensively studied tumor suppressor, regulates apoptosis and autophagy by transactivating its downstream genes. It also stimulates the mitochondrial apoptotic pathway and inhibits autophagy in a transactivation-independent manner. However, the contribution of apoptosis and autophagic cell death to p53-dependent cell death is unclear. Using wild-type (WT) and mutant (MT) p53 inducible cell lines in TP53-null SF126 glioblastoma cells, we examined the apoptosis and autophagic cell death induced by p53. WT p53 expression in SF126 cells induced apoptosis and autophagy, and reduced the cell number. An autophagy inhibitor reduced autophagy, increased the S-phase fraction, and attenuated the inhibition of cell proliferation induced by WT p53. Pan-caspase inhibitor reduced apoptosis but showed weaker inhibition of cell proliferation than the autophagy inhibitor. We concluded that p53-dependent cell death in SF126 cells comprises caspase-dependent and caspase-independent apoptosis and autophagic cell death, and the induction of autophagy as well as apoptosis could be a new strategy to treat some type of WT p53-retaining tumors.     

Constitutive phosphorylation of Janus kinase 2 in the GL15 glioblastoma derived human cell line

The notion that gliomas could originate from mutated glial precursor cells highlights the possibility of modulating the proliferative and migratory behaviour of glioma cells by acting on the molecular mechanisms operative during the development of the Central Nervous System (CNS), but absent in the normal adult brain. We show that the GL15 glioblastoma derived human cell line displays a high expression of nestin which, combined with the previously demonstrated high expression of vimentin, constitutes a characteristic of astrocyte restricted precursors. We also show that, in analogy with some leukaemia cells, GL15 cells display the constitutively phosphorylated form of Janus kinase 2 (JAK2), a tyrosine kinase expressed during CNS development but undetectable in the normal adult brain. The constitutive activation of JAK2 does not result from chromosomal aberrations involving the JAK2 gene, but most probably from abnormally activated transduction systems operative in glioblastoma cells. We then investigated the effects of tyrphostin AG490, an inhibitor of JAK2 autophosphorylation, on GL15 cell growth. In the absence of exogenous growth factors and cytokines, 10 microM tyrphostin AG490 induces an S phase arrest, combined with a partial impairment of the G2 phase of the cell cycle. The abnormally activated JAK2 could then potentially represent a target for a selective pharmacological approach in glioblastoma cells in which a combination of glial precursor characteristics and genetic alterations occurs.     

Phenotypic instability of drug sensitivity in a human colon carcinoma cell line

Colon cancer is one of the tumors most refractory to treatment by chemotherapy, and this may be due to inherent phenotypic instability of such tumor cells with respect to the biochemical determinants of drug sensitivity. To test this hypothesis, a clonal human colon carcinoma cell line, clone A, was passaged in culture in the absence of selection conditions or mutagens. During this time, sensitivity to several drugs was examined, and was found to decrease 4-fold during 30 weeks of culture. Five randomly selected subclones, having never been exposed to drug or mutagen, displayed a range of sensitivities to etoposide (50% inhibitory concentrations ranging from 1.5 to 4.9 microM) and to vincristine (9-fold range), but all had the same sensitivity to methotrexate. With time these sensitivities also changed, and subsequent subclones were chosen from the lines with highest and lowest drug sensitivity. Again a wide range of phenotypes was observed. Sensitivity to vincristine ranged 14-fold and to doxorubicin 3-fold. Several biochemical determinants of drug sensitivity had a broad range of expression between cell lines. Cellular accumulation of [3H]vincristine, as well as expression of multidrug resistance protein P170 and glutathione transferase activity all varied significantly between subclonal lines. This suggests that some human colon tumors may be phenotypically unstable with respect to drug sensitivity, and this could contribute to clinical resistance to chemotherapeutic compounds.     

Deregulated BCL2 expression enhances growth of a human B cell line

The BCL2 (B cell lymphoma/leukemia -2) and C-MYC oncogenes become activated by chromosomal translocations involving the immunoglobulin heavy chain locus in human follicular lymphomas and Burkitt lymphomas, respectively. Though much is known about the biological actions of C-MYC, little information is available concerning the functions of BCL2, particularly in human B cells. Using a gene transfer approach we contrasted the effects of deregulated BCL2 and C-MYC expression in a human EBV-immortalized B cell line GM607B. Both BCL2 and C-MYC enhanced the ability of GM607B cells to grow in reduced serum and in limiting dilution cultures. These findings provide direct evidence that BCL2 can alter the growth characteristics of human B lymphocytes, thus strengthening arguments for its role in the pathogenesis of human lymphomas.