Staurosporine differentially inhibits glioma versus non-glioma cell lines

We have previously demonstrated that the protein kinase C (PKC) activity of human glioma cell lines was significantly elevated (by 3 orders of magnitude) when compared to non-malignant adult human glia, and that the proliferation rate of several established human glioma cell lines correlated with the measured protein kinase C activity. The purpose of this study was to determine whether 1) elevated PKC activity was also a characteristic of fast growing cell lines of non-glial origin, 2) the proliferation rate of non-glioma cell lines correlated with their PKC activity and 3) the proliferation of non-glioma cell lines could be inhibited by staurosporine, a relatively selective PKC inhibitor, which significantly attenuates the growth of glioma cells. Eight established human non-glioma cell lines (bladder, colorectal, rhabdomyosarcoma-oligodendrocyte hybrid, melanoma, cervix, and fibroblast) were compared to the highly proliferative A172 glioma cell line. PKC activity was significantly higher in the glioma cell lines even though 3 of 8 of the non-glioma lines had higher proliferation rates than A172. In non-glioma cell lines, no correlation was found between the PKC activity and proliferation rates. Staurosporine was more effective in decreasing the proliferation of three glioma cell lines compared to the non-glioma cell lines. We conclude that PKC activity is differentially increased in glioma cell lines and that their proliferation rate is more sensitive to inhibition by Staurosporine. Targetting the PKC system may prove to be of therapeutic benefit to patients with malignant gliomas.     

A comparison of the relative chemosensitivity of human gliomas to tamoxifen and n-desmethyltamoxifenin vitro

Summary Tamoxifen has been shown to inhibit the proliferation of human gliomasin vitro. This inhibition is independent of tamoxifen's known anti-estrogenic properties. Tamoxifen is an inhibitor of protein kinase C (PKC), a calcium- and phospholipid-dependent serine kinase which plays a critical role in the proliferation of certain cell lines. Gliomas overexpress PKC, and their growth rate is coupled to the level of this key enzyme. As such, the effect of tamoxifen may be mediated by its inhibitory effect on PKC. To further investigate this possibility, we compared the chemosensitivity of cultured glioma lines to both tamoxifen and N-desmethyltamoxifen (DMT). DMT is the major metabolite of tamoxifen in humans and is a ten-fold more potent inhibitor of PKC. Seven lines were tested using the standard MTT assay, which quantitates metabolically active cells colorimetrically using a tetrazolium dye. Four of the seven lines were also tested using a tritiated thymidine uptake assay. In the MTT assay, all seven lines showed significantly greater sensitivity to DMT, while three of the four lines tested in the thymidine uptake assay were more sensitive to DMT. Correlation between the two assays was good. The dose of tamoxifen required to produce a 50% inhibition of optical absorbance or thymidine uptake (ID50) was typically five- to ten-fold greater than the ID50 for DMT, approximating the relative strength of the two compounds as PKC inhibitors. In addition to providing some support for the ypothesis that triphenylethylenes inhibit gliomas via PKC inhibition, these findings have clinical significance. Levels of DMT in serum, brain and brain metastases are about twice the comparable levels of tamoxifen during chronic therapy. DMT achieved complete inhibition of all seven glioma lines at concentrations of 200 ng/ml. Levels as high as 2000 ng/g can be achieved in tumor tissue, suggesting that chronic tamoxifen therapy may have some role in the therapy of these aggressive tumors.     

Ribozyme inhibition of the protein kinase C alpha triggers apoptosis in glioma cells.

Although protein kinase C has been shown to be involved in a wide range of biological functions, the precise role of each isoform in a specific cell function remains to be clarified. Here we demonstrate that a ribozyme specific for the human protein kinase C alpha (PKC alpha), a classical PKC isoform, induces cell death in glioma cell lines. This cell death was identified as apoptosis by morphologic alterations and endonucleosomal DNA fragmentation. The inhibition of PKC alpha gene expression by the ribozyme resulted in a significant reduction in Bcl-xL gene expression, a protein that inhibits apoptosis and is overexpressed in glioma cells. Taken together, our data suggest that the PKC alpha ribozymes are a potent inducer of apoptosis in glioma cells, which may act through suppressing Bcl-xL gene expression and/or activity. PKC alpha ribozymes may prove useful in the management of malignant gliomas.     

Effect of tamoxifen on DNA synthesis and proliferation of human malignant glioma lines in vitro

Previous studies in our laboratory have shown that proliferation of human malignant gliomas in vitro depends in part upon the activation of protein kinase C (PKC) and, conversely, can be blocked by inhibitors of PKC. Here, we examined the effect of tamoxifen, a known PKC inhibitor, on DNA synthesis and proliferation of an established human glioma line (U138) and two low passage cultures of explanted human glioblastomas. Tamoxifen produced a profound, dose-dependent inhibition of both [3H] thymidine incorporation and cell proliferation, with a 50% effective dose of 20 ng/ml under serum-free conditions and 50 to 200 ng/ml in the presence of 10% serum. These tumors were estrogen receptor negative and showed no mitogenic response to estradiol. Furthermore, concentrations of estradiol as high as 10 micrograms/ml had no effect on the tamoxifen-induced inhibition. This suggests that the mechanism of growth inhibition by tamoxifen in these gliomas did not involve an estrogen receptor-mediated process but may instead result from its inhibition of PKC. In view of the profound effect of tamoxifen on cultured gliomas at concentrations that can safely be achieved therapeutically, further in vitro and in vivo studies of this agent are warranted.     

PKC alpha protein but not kinase activity is critical for glioma cell proliferation and survival

Protein kinase C alpha (PKCalpha) has been implicated in tumor development with high levels of PKCalpha expression being associated with various malignancies including glioblastomas and tumors of the breast and prostate. To account for its upregulation in these cancers, studies have suggested that PKCalpha plays a role in promoting cell survival. Here we show by siRNA depletion in U87MG glioma cells that a critical threshold level of PKCalpha protein expression is essential for their growth in the presence of serum and for their survival following serum deprivation. Derivation of PKCalpha wt and KO mouse embryo fibroblast cell lines confirms a role for PKCalpha in protecting cells from apoptosis induced by serum deprivation. Notably, PKCalpha was found to mediate chemo-protection in these fibroblastic cell lines. In U87MG cells PKCalpha does not confer chemoprotection though this likely reflects growth arrest associated with its depletion. To determine the requirements for catalytic function, comparison was made between distinct classes of PKC inhibitors. In contrast to loss of PKCalpha protein, inhibition of PKC kinase activity in glioma cell lines does not significantly inhibit growth or survival. Conversely, inhibition with calphostin C, which targets the regulatory domain of PKC, potently inhibits proliferation and induces apoptosis. Evidence is presented that it is the fully phosphorylated, folded form of PKCalpha that confers this activity-independent behaviour. These results indicate an essential pro-proliferative and pro-survival role for PKCalpha in glioma but question the use of ATP competitive inhibitors as therapeutics, either alone, or in combination with chemotoxic agents.     

血管生成抑制素对C6脑胶质瘤的抑瘤效应

目的探讨不同剂量的血管生成抑制素（Angiostatin）在胶质瘤治疗中的抑制血管生成作用。方法应用不同剂量（100ng、1000ng）的血管生成抑制素分别作用于体外培养的大鼠C6脑胶质瘤细胞系和C6/SD大鼠脑胶质瘤模型,分别计算细胞存活率和抑瘤生成率,SABC免疫组织化学技术检测体内胶质瘤中的微血管密度。结果不同剂量的血管生成抑制素均对体外培养的胶质瘤细胞的生长不产生明显抑制作用;对体内生长的胶质瘤则有显著抑制作用,而且较高剂量的Angiostatin的抑制作用更为明显,抑瘤率最高达65.3％（P<0.01）。经不同剂量的血管生成抑制素处理的脑胶质瘤中微血管密度较对照组降低（P<0.01）。结论血管生成抑制素能抑制C6脑胶质瘤的血管生成,且较高剂量实验组的作用更为明显,此研究对血管生成抑制素抑制C6胶质瘤的生长机制研究奠定了一定基础。     

The heat shock protein antagonist 17-AAG potentiates the activity of enzastaurin against malignant human glioma cells

Recent studies have suggested that the proliferation of malignant gliomas may result from activation of protein kinase C (PKC)-mediated pathways. Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of PKCbeta as well as other isoforms. The initial objective of this study was to assess the efficacy of enzastaurin in a series of malignant human glioma cell lines with diverse genomic alterations. Although enzastaurin independently produced a dose-dependent inhibition of cellular proliferation and decreased cell viability in each of the glioma cell lines examined, and partially down-regulated Akt and GSK3beta phosphorylation, median effective concentrations were at the upper limits of, or above, the clinically achievable range in all cell lines tested. We therefore examined whether the efficacy of enzastaurin could be enhanced by combination with the HSP90 antagonist, 17-AAG, which inhibits Akt and other signaling intermediates by a distinct mechanism. In comparison to the effect of enzastaurin alone, combination of enzastaurin with 17-AAG led to marked enhancement of antiproliferative and cytotoxic effects. Simultaneous exposure to both agents significantly increased the release of cytochrome c, as well as caspase 3 activation, Bax cleavage, and inhibition of Akt phosphorylation. Cells exposed to enzastaurin and 17-AAG also displayed a significant reduction in cell cycle regulatory proteins, such as CDK4 and CDK6. Taken together, these findings suggest that the efficacy of enzastaurin can be potentiated by the addition of 17-AAG, and indicate that combining molecularly targeted therapies may provide a more effective strategy than single-agent therapy to treat patients with malignant gliomas.     

The topoisomerase II inhibitor, genistein, induces G2/M arrest and apoptosis in human malignant glioma cell lines

The protein tyrosine kinase inhibitor, genistein, has been reported to inhibit proliferation and to induce cell death in various non-solid and solid cancer cell lines. Herein, we examined the effects of genistein in several human malignant glioma cell lines. We found that genistein inhibited the proliferation of LN-18, LNT-229, LN-308 and T98G cells at EC50 concentrations of 25-80 microM (72 h of exposure). The growth of a non-neoplastic immortalized human astrocyte cell line, SV-FHAS, was inhibited at similar concentrations. There was a reduction in [3H]-methylthymidine incorporation and a moderate lactate dehydrogenase release as a sign of cell death in genistein-treated glioma cells. Electron microscopy showed morphological changes with mitochondrial swelling and apoptosis in glioma cells treated with high concentrations of genistein. Genistein-induced cytotoxicity was associated with an increased DNA/topoisomerase II complex formation. Furthermore, genistein induced cell cycle arrest in G2/M. There was an increase in the p53 and p21 levels in response to genistein. However, there was no difference in genistein sensitivity between p21-deficient colon carcinoma cells and isogenic control cells. Genistein-induced cell death in LN-18 and LNT-229 was unaffected by the ectopic expression of the preferential caspase 1/8 inhibitor, crm-A, or co-exposure to the pan-specific pseudosubstrate caspase inhibitor, zVAD-fmk. The ectopic expression of the anti-apoptotic BCL-2 protein attenuated the cytotoxic effects of genistein. Moreover, the ectopic expression of temperature-sensitive p53V135A, which acts as a dominant-negative p53 mutant at 38.5 degrees C but assumes p53 wild-type properties at 32.5 degrees C, in LN-18 or LNT-229 cells, had no effect on genistein cytotoxicity at either temperature. Genistein did not act in synergy with CD95 ligand-induced apoptosis or various cancer chemotherapy drugs in cytotoxic or clonogenic cell death assays. Thus, genistein-like protein kinase inhibitors are promising agents for the experimental treatment of malignant gliomas.     

Protein kinase C zeta mediates epidermal growth factor-induced growth of head and neck tumor cells by regulating mitogen-activated protein kinase

Protein kinase C (PKC) zeta has been implicated as a mediator of epidermal growth factor (EGF) receptor (EGFR) signaling in certain cell types. Because EGFR is ubiquitously expressed in squamous cell carcinomas of the head and neck (SCCHN) and plays a key role in tumor progression, we determined whether PKCzeta is required for tumor cell proliferation and viability. Examination of total and phosphorylated PKCzeta expression in normal oral mucosa, dysplasia, and carcinoma as well as SCCHN tumor cell lines revealed a significant increase in activated PKCzeta expression from normal to malignant tissue. PKCzeta activity is required for EGF-induced extracellular signal-regulated kinase (ERK) activation in both normal human adult epidermal keratinocytes and five of seven SCCHN cell lines. SCCHN cells express constitutively activated EGFR family receptors, and inhibition of either EGFR or mitogen-activated protein kinase (MAPK) activity suppressed DNA synthesis. Consistent with this observation, inhibition of PKCzeta using either kinase-dead PKCzeta mutant or peptide inhibitor suppressed autocrine and EGF-induced DNA synthesis. Finally, PKCzeta inhibition enhanced the effects of both MAPK/ERK kinase (U0126) and broad spectrum PKC inhibitor (chelerythrine chloride) and decreased cell proliferation in SCCHN cell lines. The results indicate that (a) PKCzeta is associated with SCCHN progression, (b) PKCzeta mediates EGF-stimulated MAPK activation in keratinocytes and SCCHN cell lines, (c) PKCzeta mediates EGFR and MAPK-dependent proliferation in SCCHN cell lines; and (d) PKCzeta inhibitors function additively with other inhibitors that target similar or complementary signaling pathways.     

Combination effect of hyperthermia and MX2 on cultured glioma cell lines]

MX2, a new lipophilic morpholino anthracycline, has been reported to have chemotherapeutic effects superior to those of adriamycin against murine and human glioma cells in vitro and in vivo. To assess the combination effect of MX2 and hyperthermia in vitro, the thermo-chemosensitivities of cultured human (U251MG and KC) and rat (C6 and 9L) glioma cell lines were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. The number of viable cells in each cell line was markedly and dose-dependently decreased by MX2 treatment, but the sensitivity of U251MG to MX2 was less than that of the other cell lines. The survival of each cell line was decreased with the hyperthermic treatment at 43 degrees C for 60 minutes. Combined treatment with MX2 and hyperthermia had an additive effect on cultured glioma cells when MX2 was added to the medium at a dose below 50 ng/ml. However, combined treatment indicated neither an additive nor a synergistic effect when the dose of MX2 was above 50 ng/ml. We conclude that MX2 may be clinically useful against malignant gliomas when administered alone or in combination with hyperthermia.     

Direct stimulation of apoptotic signaling by soluble Apo2l/tumor necrosis factor-related apoptosis-inducing ligand leads to selective killing of glioma cells.

Apo2 ligand tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the tumor necrosis factor family that interacts with cell surface "death receptors" (DR4 and DR5) to initiate programmed cell death. Apo2L/TRAIL also binds to "decoy" receptors (DcR1 and DcR2) that can antagonize its interaction with DR4 and DR5. In recent studies, Apo2L/TRAIL has been noted to produce selective toxicity toward certain neoplastic cells versus normal cells. The decoy receptors may in part contribute to this selectivity, because they are expressed in various normal tissues but are present at low or undetectable levels in certain types of neoplastic cells. In the current study, we examined the potential therapeutic applicability of recombinant soluble Apo2L/TRAIL by investigating its effects in vitro and in vivo against a series of cell lines derived from malignant gliomas, which are often resistant to conventional treatment modalities. In cell proliferation assays, Apo2L/TRAIL produced a striking decrease in cell numbers, with a median inhibitory concentration of 30-100 ng/ml, in the TP53 wild-type high-grade glioma cell lines U87 and A172, the TP53-mutated T98G, and the TP53-deleted LN-Z308. In contrast, no significant effects were observed in non-neoplastic astrocytes at concentrations up to 3000 ng/ml. Clonogenic assays showed that exposure to Apo2L produced a time-dependent decrease in the viability of glioma-derived cell lines. This correlated with the induction of apoptosis as assessed by a terminal transferase-catalyzed in situ end-labeling assay. Pretreatment of the cells with the caspase inhibitors Acetyl-Asp-Glu-Val-L-aspartic acid aldehyde or Acetyl-Tyr-Val-Ala-Asp-chlormethylketone (200 microM) largely eliminated the effects of Apo2L/TRAIL. Administration of Apo2L/TRAIL (0.3, 1, 3, 10, and 30 mg/kg/day for 7 days via i.p. infusion) to nude mice harboring established intracranial U87 xenografts produced a significant, dose-dependent prolongation of survival versus control animals. Survival in the control group was 27 +/- 1.7 days, compared with more than 50 days in each of the treatment groups (P < 0.001). At the 30 mg/kg dose level, 100% of animals survived for 120 days without evidence of tumor, a substantial improvement in comparison with lower dose levels (P < 0.01). No overt toxicity was apparent even at the highest Apo2L dose. We conclude that soluble Apo2L/TRAIL is effective in inducing apoptosis in high-grade glioma cells in vitro. Because this ligand appears to exhibit selective cytotoxicity for glioma cells versus non-neoplastic cells in vitro and demonstrates significant activity in vivo when administered systemically in an otherwise uniformly fatal central nervous system glioma model system, Apo2L may constitute a useful therapeutic agent for these challenging tumors.     

Inhibitory effect of epigallocatechin-gallate on brain tumor cell lines in vitro

We investigated the effect of epigallocatechin-gallate (EGCG), the main constituent of green tea polyphenols, on human glioblastoma cell lines U-373 MG and U-87 MG, rat glioma cell line C6, and rat nonfunctioning pituitary adenoma cell line MtT/E. Cell viability was determined by assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and the extent of apoptosis was studied by flow cytometric analysis. Apoptosis was also characterized by morphology using fluorescent microscopy. The role of insulin-like growth factor-I (IGF-I) was studied by assay with MTT, immunohistochemistry, and immunoradiometric assay. After 72-h exposure, a statistically significant loss of viability (P = < 0.0001) was observed at concentrations of 12.5, 25, 50, and 100 microg/ml in U-373 MG cells and U-87 MG cells. EGCG at concentrations of 50 microg/ml and higher significantly reduced the viability of C6 cells. EGCG inhibited viability of MtT/E cells only at a concentration of 100 microg/ml. Quantitative study by flow cytometry demonstrated that lower doses of EGCG (12.5, 25, 50 microg/ml) induced apoptosis in U-373 MG, U-87 MG, and C6 cells; however, only the highest dose (100 microg/ml) induced apoptosis in MtT/E cells. Compared with other cell lines, MtT/E cells showed stronger IGF-I immunoreactivity. Neutralization of IGF-I with an antihuman IGF-I antibody reduced viability of the cell lines. It can be concluded that EGCG has an inhibitory effect on malignant brain tumors, and IGF-I may be involved in the effects of EGCG.     

Potent Killing of Paclitaxel- and Doxorubicin-resistant Breast Cancer Cells by Calphostin C Accompanied by Cytoplasmic Vacuolization

Drug resistance is a major impediment to the successful treatment of breast cancer using chemotherapy. The photoactivatable drug calphostin C has shown promise in killing select drug-resistant tumor cells lines in vitro. To assess the effectiveness of this agent in killing doxorubicin- or paclitaxel-resistant breast tumor cells and to explore its mode of action, MCF-7 cells were exposed to increasing concentrations of either doxorubicin or paclitaxel until maximum resistance was obtained. This resulted in the creation of isogenic drug-resistant MCF-7TAX and MCF-7DOX cell lines, which were approximately 50- and 65-fold resistant to paclitaxel and doxorubicin, respectively. Interestingly, calphostin C was able to kill MCF-7TAX cells as efficiently as wildtype MCF-7 cells (IC50s were 9.2 and 13.2 nM, respectively), while MCF-7DOX cells required a 5-fold higher concentration of calphostin C to achieve the same killing (IC50 = 64.2 nM). Consistent with their known mechanisms of action, paclitaxel killed tumor cells by inducing mitotic arrest and cell multinucleation, while doxorubicin induced plasma membrane blebbing and decreased nuclear staining with propidium iodide. In contrast, cytoplasmic vacuolization accompanied cell killing by calphostin C in these cell lines, without the induction of caspase-8 or PARP cleavage or the release of cytochrome c from mitochondria. Calphostin C had little effect on the uptake of either paclitaxel or doxorubicin by the cells. Taken together, the above data suggests that calphostin C is able to potently kill drug-resistant breast tumor cells through a mechanism that may involve the induction of cytoplasmic vacuolization, without activation of typical apoptotic pathways. Consequently, calphostin C may prove useful clinically to combat tumor growth in breast cancer patients whose tumors have become unresponsive to anthracyclines or taxanes, particularly in association with photodynamic therapy.     

Sensitization of C6 glioma cells to radiation by staurosporine,a potent protein kinase C inhibitor

Summary The effect of staurosporine, a potent protein kinase C (PKC) inhibitor, on the sensitivity to radiation has been investigated in C6 glioma cells. Pretreatment of C6 cells with staurosporine at the concentrations over 1 nM resulted in an enhancement of sensitivity to irradiation. At a concentration of 5 nM, staurosporine caused significant radiosensitization of the cells, either it was administered 1) before and during irradiation, or 2) continuously before, during, and after irradiation, with a reduced D₀ (the 37% survival dose) from 3.8 Gy to 2.9 Gy and 3.0 Gy, respectively, (p< 0.03). Since the viability of C6 cells was not affected by staurosporine alone at the concentrations tested, the radiosensitizing effect of staurosporine was considered to be mediated via suppression of PKC. Furthermore, another potent PKC inhibitor H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, also sensitized C6 cells to irradiation, while HA1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride a potent inhibitor for cAMP-dependent protein kinase, failed to affect the radiosensitivity in this cells. Therefore, staurosporine-induced sensitization of C6 cells to radiation may at least in part be mediated by its inhibitory activity for PKC. Staurosporine represents a new agent for radiosensitization and may prove usefulness in studying the mechanisms responsible for radio-resistance and -sensitivity in glioma cells.     

Differential phorbol ester growth modulation and protein-kinase-C isoenzyme expression in rat hepatoma-cells vs non neoplastic rat hepatocytes

In the present study the expression of protein kinase C (PKC) isoenzymes and the growth modulation caused by phorbol esters were studied in two rat hepatic cell lines: the neoplastic MH1C1 hepatoma and the syngeneic immortalized non-neoplastic BRL3A cell line. Western blot analysis revealed that MH1C1 rat hepatoma cells express the a isoenzyme as the only isoform of PKC, whereas BRL3A cells showed immunoreactivity for alpha, delta and epsilon PKC. Immunoblot analysis showed that in both cell lines phorbol 12-myristate 13-acetate (PMA) caused a persistent, marked down-regulation of all expressed PKC isoenzymes. In MH1C1 cells, PMA also induced a reversible, marked dose-dependent growth inhibition, whereas a minimal impairment of cell proliferation was observed in BRL cells. The observed selective mitoinhibitory effect of PMA on tumor cells indicates PKC as an interesting target for innovative antiproliferative strategies based on the modulation of specific isoenzymes.     

Deletion analysis of protein kinase c inactivation by calphostin C

Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC-α (a Ca²⁺-dependent conventional isoform) and PKC-α (a Ca²⁺-independent novel isoform) are similarly inactivated by calphostin C (75–100 nM produced 50% inhibition), suggesting that inactivation requires a site common to both classes of PKC. We therefore performed studies to identify a critical region in the regulatory domain of PKC-α required for inactivation by calphostin C. A series of N-terminal–truncation mutants of bovine PKC-α expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild-type PKC-α by 80–90%. This concentration was as effective with mutant proteins containing deletions of up to 91 amino acid (aa) residues from the amino terminus (ND91), whereas a mutant protein truncated by 140 aa (ND 140) was inactivated by only 20%. These findings imply that the aa sequence 92–140 is a structural determinant of PKC-α inactivation by calphostin C. This sequence contains one of the phorbol ester-binding sites (aa 102–144), which is highly conserved among most PKC isoforms including PKC-α. In addition to aa 92–140, PKC-stimulating cofactors (phosphatidylserine, phorbol ester, and Ca²⁺) are required for inactivation by calphostin C even in the case of PKC mutants that do not require these cofactors for enzymatic activity. These results suggest that cofactors provide a template that is required for productive interaction of PKC and the inhibitor. The significance of the proposed proximity effect of calphostin C action is discussed. © 1995 Wiley-Liss Inc.     

Aurora kinase B/C inhibition impairs malignant glioma growth in vivo

Inhibition of Aurora kinase B has been evaluated as a therapy to block solid tumor growth in breast cancer, hepatocellular carcinoma, lung adenocarcinoma, and colorectal cancer models. Aurora kinase inhibitors are in early clinical trials for the treatment of leukemia. We hypothesized that Aurora B inhibition would reduce malignant glioma cell viability and result in impaired tumor growth in vivo. Aurora B expression is greater in cultured malignant glioma U251 cells compared to proliferating normal human astrocytes, and expression is maintained in U251 flank xenografts. Aurora B inhibition with AZD1152-HQPA blocked cell division in four different p53-mutant glioma cell lines (U251, T98G, U373, and U118). AZD1152-HQPA also inhibited Aurora C activation loop threonine autophosphorylation at the effective antiproliferative concentrations in vitro. Reduction in cell viability of U251 (p53(R273H)) cells was secondary to cytokinesis blockade and apoptosis induction following endoreplication. AZD1152-HQPA inhibited the growth of U251 tumor xenografts and resulted in an increase in tumor cell apoptosis both in vitro and in vivo. Subcutaneous administration of AZD1152-HQPA (25 mg/kg/day × 4 days; 2 cycles spaced 7 days apart) resulted in a prolongation in median survival after intracranial inoculation of U251 cells in mice (P = 0.025). This is the first demonstration that an Aurora kinase inhibitor can inhibit malignant glioma growth in vivo at drug doses that are clinically relevant.     

蛋白激酶C抑制剂Staurosporine对人胃癌细胞周期的影响

背景与目的:蛋白激酶C(proteinkinaseC,PKC)已成为一个潜在的有价值的抗肿瘤治疗靶点。本研究旨在探讨PKC抑制剂Staurosporine(ST)对人胃癌细胞株MGC-803和SGC-7901的增殖抑制、凋亡诱导及对其细胞周期的影响。方法:在用台盼蓝拒染法检测细胞增殖抑制率的基础上,通过细胞形态学观察凋亡小体,流式细胞仪检测细胞凋亡率及细胞周期变化。结果:ST抑制MGC-803细胞生长,24h的IC50为54ng/ml,48h的IC50为23ng/ml;ST抑制SGC-7901细胞的生长,24h的IC50为61ng/ml,48h的IC50为37ng/ml。40、60、100ng/mlST分别作用MGC-803细胞24h后,发现G0/G1期细胞分别为(23.6±1.8)%、(11.6±0.7)%、(3.3±0.2)%,而对照组为(54.3±3.1)%;G2/M期细胞分别为(22.6±4.0)%、(35.5±0.4)%、(36.8±5.5)%,而对照组为(13.5±0.2)%。40、60、100ng/mlST分别作用于SGC-7901细胞24h后,G0/G1期细胞分别为(27.1±1.4)%、(17.0±3.4)%,(13.7±0.7)%,而对照组为(52.5±4.4)%;G2/M期细胞分别为(21.9±2.6)%、(39.5±4.9)%、(38.4±3.1)%,而对照组为(13.5±2.2)%。实验组细胞与对照组比较,ST使两种细胞G0/G1期明显减少及G2/M期明显增加(P<0.01)。200ng/mlST作用24h后两种细胞的G1期前均出现明显的凋亡峰,可诱导细胞出现典型凋亡小体。结论:ST显著降     

Selection and preliminary characterization of variant lines of a murine macrophage tumor resistant to the antiproliferative effects of phorbol esters

Treatment of M5076 wild-type cells with 50 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) almost completely inhibited cellular proliferation. Continuous culture in the presence of TPA was used to derive four lines, one polyclonal (TPAR) and three clonally derived (TPAR-1, -2, and -3), which exhibited variable resistance to the antiproliferative effects of phorbol esters. Protein kinase C (PKC) activation and c-fos expression in wild-type cells and the stably resistant line (TPAR-3) were examined after phorbol ester treatment. Both lines exhibited a comparable rapid and transient induction of c-fos mRNA expression, but induction of c-fos protein was reduced markedly in the TPAR-3 cells. Similarly in both cell lines, prolonged culture in phorbol ester produced down-regulation of PKC, as measured by inducible Mr 80,000 phosphorylation and an in vitro PKC assay. This decrease in PKC levels was paralleled by a decrease in c-fos mRNA and protein induction. Thus, c-fos expression in both wild-type and TPAR-3 cells is a consequence of PKC activation, and the development of resistance to TPA-antiproliferative effects in the TPAR-3 cell line was not linked causally to alterations in PKC levels or the c-fos mRNA induction response. The malignant capacity of the TPAR line was not reduced relative to wild-type cells. PKC activation and c-fos mRNA expression do not appear to determine changes in the in vivo or in vitro growth behavior of M5076 cells, whereas variations in c-fos protein expression may determine the anti-proliferative response to tumor-promoting phorbol esters.     

Protein kinase C isozyme expression in human leukemia-lymphoma cell lines--an immunocytochemical study.

There have been an increasing number of reports describing a pivotal role for phosphorylation in cellular responses for cell differentiation and proliferation. We examined an immunocytochemical expression of protein kinase C(PKC) isozymes (type I, II, and III) in 22 leukemia-lymphoma cell lines. Of these cell lines, 21 expressed type II PKC and 17 showed the co-expression of both types II and III PKC in varying degree. The cell line without PKC activity showed far less [3H]-TdR uptake and no heterotransplantation in nude mice. Types II and III PKC appear to relate to cell proliferation in certain leukemia-lymphoma cell lines.