Role of VEGF and matrix metalloproteinase-9 in peritumoral brain edema associated with supratentorial benign meningiomas.

Accumulating evidence indicates that VEGF and matrix metalloproteinase-9 (MMP-9) play a central role in the development of peritumoral brain edema (PTBE) associated with human brain tumors. However, the roles of these proteins, particularly of MMP-9, in PTBE associated with benign meningiomas have not been elucidated. We investigated the association between clinical features and biological factors, such as VEGF and MMP-9, and the incidence of PTBE and edema index (EI) in 60 patients with benign meningiomas. In this study, supratentorial lesions were examined for evaluating the extent of PTBE in the surrounding normal brain tissue. VEGF and MMP-9 expression was immunohistochemically examined. Multivariate analysis revealed that the presence of pial blood supply (odds ratio [OR] 12.250; P = 0.0096) and VEGF (OR 7.683; P = 0.0155), but not MMP-9 (OR 1.178; P = 0.8113), expression are significant factors that independently predict the incidence of PTBE and influence EI. VEGF (P = 0.0397) and MMP-9 (P = 0.0057) expression correlates with the presence of pial blood supply. Moreover, tumors with high VEGF and MMP-9 expression had higher EIs than those with high expression of either (P = 0.030). Our findings suggest that MMP-9 expression was positively related to VEGF expression and pial blood supply and promoted the occurrence of PTBE by inducing the disruption of the arachnoid membrane and formation of pial blood supply.

Temsirolimus downregulates p21 without altering cyclin D1 expression and induces autophagy and synergizes with vorinostat in mantle cell lymphoma.

OBJECTIVE: To investigate the mechanisms of antiproliferative effect induced by the mammalian target of rapamycin (mTOR) inhibitor temsirolimus in mantle cell lymphoma (MCL). MATERIALS AND METHODS: The antiproliferative effect of temsirolimus on three well-defined MCL cell lines was examined by the MTS assay. Induction of cell-cycle arrest, autophagy, and apoptosis were determined by fluorescence-activated cell sorting analysis. The molecular mechanisms underlining these effects were determined by Western blot. Synergy between temsirolimus and vorinostat were examined by MTS assay and the combination index was calculated. RESULTS: Temsirolimus has antiproliferative activity in three MCL cell lines in a dose- and time-dependent manner. Mechanistically, temsirolimus inhibited mTOR, as evidenced by inhibition of ribosomal S6 phosphorylation, and induced cell-cycle arrest in the G(0)/G(1) phase and a decrease in p21 expression without altering p27 or cyclin D1 levels. Furthermore, temsirolimus increased the number of acidic vesicular organelles and the amount of microtubule-associated protein 1 light-chain 3 processing, which are characteristic of autophagy, without induction of apoptosis. These changes were not associated with alteration in phosphorylated extracellular signal-regulated kinase (ERK), beclin-1, Bax, or Bak levels. In contrast, treatment of these cell lines with the histone deacetylase inhibitor vorinostat decreased ERK phosphorylation, activated caspase 3, and induced apoptosis. Moreover, temsirolimus synergized with submaximal concentrations of vorinostat in all MCL cell lines. CONCLUSION: This is the first report of temsirolimus-induced autophagy in MCL, and of vorinostat inhibition of ERK phosphorylation in MCL. Collectively, these data suggest that the combination of temsirolimus and vorinostat have synergistic antiproliferative activity in MCL cells by distinctively targeting apoptosis and autophagy.

Monitoring autophagy in glioblastoma with antibody against isoform B of human microtubule-associated protein 1 light chain 3.

Autophagy, an evolutionarily conserved response to stress, has recently been implicated in cancer initiation and progression, but the detailed mechanisms and functions have not yet been fully elucidated. One major obstacle to our understanding is lack of an efficient and robust method to specifically monitor autophagic cells in cancer specimens. To identify molecular events associated with autophagy, we performed cDNA microarray analysis of autophagic glioblastoma cell lines. Based on the analysis, we raised a polyclonal antibody against isoform B of human microtubule-associated protein 1 light chain 3 (LC3B). Application of the anti-LC3B antibody revealed the presence of autophagic cells in both in vitro and in vivo settings. Of the 65 glioblastoma tissues, 31 had highly positive cytoplasmic staining of LC3B. The statistical interaction between cytoplasmic staining of LC3B and Karnofsky Performance Scale score was significant. High expression of LC3B was associated with an improved outcome for patients with poorer performance, whereas, for patients with normal performance, survival was better for patients with low staining than with high staining of LC3B. Anti-LC3B antibody provides a useful tool for monitoring the induction of autophagy in cancer cells and tissues.

Pivotal role of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in apoptosis and autophagy.

Programmed cell death (PCD) is involved in a variety of biologic events. Based on the morphologic appearance of the cells, there are two types of PCD as follows: apoptotic (type I) and autophagic (type II). However, the molecular machinery that determines the type of PCD is poorly defined. The purpose of this study was to show whether the presence of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1), a modulator of apoptosis, determines which type of PCD the cell undergoes. Treatment with C(2)-ceramide was associated with both the cleavage of caspase-3 and poly(ADP-ribose) polymerase and the degradation of autophagy-related Beclin 1 and Atg5 proteins, without a change in the cyclin-CDK activity, which culminated in apoptosis in p21(+/+) mouse embryonic fibroblasts (MEFs). On the other hand, C(2)-ceramide did not cleave caspase-3 or poly(ADP-ribose) polymerase and kept Beclin 1 and Atg5 proteins stable in p21(-/-) MEFs, events that this time culminated in autophagy. When expression of the p21 protein was inhibited by small interfering RNA or when the overexpression of Beclin 1 or Atg5 was induced, autophagy rather than apoptosis was initiated in the p21(+/+) MEFs treated with C(2)-ceramide. In contrast, the exogenous expression of p21 or the silencing of Beclin 1 and Atg5 with small interfering RNA increased the number of apoptotic cells and decreased the number of autophagic cells among C(2)-ceramide-treated p21(-/-) MEFs. gamma-Irradiation, which endogenously generates ceramide, induced a similar tendency in these MEFs. These results suggest that p21 plays an essential role in determining the type of cell death, positively for apoptosis and negatively for autophagy.

Combined effect of 2-5A-linked antisense against telomerase RNA and conventional therapies on human malignant glioma cells in vitro and in vivo.

We recently showed that therapy with 2'-5'-oligoadenylate (2-5A)-linked antisense against human telomerase RNA component (2-5A-anti-hTR) is a novel telomerase-targeting strategy against malignant gliomas. In this study, we investigated conventional chemotherapeutic agents and gamma-irradiation (IR) to determine whether they could augment the efficacy of 2-5A-anti-hTR against these tumors in vitro and in vivo. Treatment with 2-5A-anti-hTR inhibited the viability of U373-MG and U87-MG malignant glioma cells in a dose-dependent manner; the antitumor effect resulted from induction of apoptosis. Also, telomerase-positive astrocytes with oncogenic Ras were more sensitive to 2-5A-anti-hTR than were those without oncogenic Ras. In addition, we sought to determine the combined effect of 2-5A-anti-hTR with N, N'-bis (2-chloroethyl)-N-nitrosourea (BCNU), cisplatin (CDDP), paclitaxel (PTX), temozolomide (TMZ), or IR. When we administered the combination treatments on the same day, PTX and IR showed a greater combined effect with 2-5A-anti-hTR on both tumor cell lines than did BCNU, CDDP and TMZ. However, all of the combination regimens were synergistic when we first treated tumor cells with 2-5A-anti-hTR for 24 h and then exposed them to the conventional treatments. Apoptosis-inducing agents (CDDP and PTX) but not autophagy-inducing therapies (TMZ and IR) enhanced the incidence of apoptosis caused by 2-5A-anti-hTR. Lastly, we observed a combinatorial effect of 2-5A-anti-hTR and TMZ in vivo in subcutaneous U87-MG tumors in nude mice. Interestingly, treatment with TMZ increased the incidence of apoptosis in subcutaneous tumor cells treated with 2-5A-anti-hTR. These results suggest that 2-5A-anti-hTR is preferable in combination with established cancer therapies.

Akt inhibitor shows anticancer and radiosensitizing effects in malignant glioma cells by inducing autophagy.

Autophagy, or programmed cell death type II, is one of the responses of cancer cells to various therapies, including ionizing radiation. Recently, we have shown that radiation induces autophagy, but not apoptosis, in various malignant glioma cell lines. Autophagy is mainly regulated by the mammalian target of rapamycin (mTOR) pathway. The Akt/mTOR pathway also mediates oncogenesis and radioresistance. Thus, we hypothesized that inhibiting this pathway has both an anticancer and radiosensitizing effect by activating autophagy. The purpose of our study was therefore to determine whether and by which mechanisms an Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate, had anticancer and radiosensitizing effects on malignant glioma U87-MG and radioresistant U87-MG cells with a consistitutively active form of epidermal growth factor receptor (U87-MGDeltaEGFR). Treatment with the Akt inhibitor successfully inhibited Akt activity and reduced cell viability in both cell lines. In terms of the mechanism, the Akt inhibitor decreased phosphorylated p70S6 kinase, a downstream target of Akt, and induced autophagy, but not apoptosis. Furthermore, the Akt inhibitor radiosensitized both U87-MG and U87-MGDeltaEGFR cells by enhancing autophagy. Specific inhibition of Akt using the dominant-negative Akt plasmid also resulted in enhanced radiation-induced autophagy. In conclusion, an Akt inhibitor showed anticancer and radiosensitizing effect on U87-MG and U87-MGDeltaEGFR cells by inducing autophagy. Thus, Akt inhibitors may represent a promising new therapy as a single treatment or used in combination with radiation for malignant gliomas, including radioresistant ones that express DeltaEGFR.

Telomere 3' overhang-specific DNA oligonucleotides induce autophagy in malignant glioma cells.

Telomere 3' overhang-specific DNA oligonucleotides (T-oligos) induce cell death in cancer cells, presumably by mimicking telomere loop disruption. Therefore, T-oligos are considered an exciting new therapeutic strategy. The purpose of this study was to elucidate how T-oligos exert antitumor effects on human malignant glioma cells in vitro and in vivo. We demonstrated that T-oligos inhibited the proliferation of malignant glioma cells through induction of nonapoptotic cell death and mitochondria hyperpolarization, whereas normal astrocytes were resistant to T-oligos. Tumor cells treated with T-oligos developed features compatible with autophagy, with development of autophagic vacuoles and conversion of an autophagy-related protein, microtubule-associated protein 1 light chain 3 from type I (cytoplasmic form) to type II (membrane form of autophagic vacuoles). A reverse-phase protein microarray analysis and Western blotting revealed that treatment with T-oligos inhibited the mammalian target of the rapamycin (mTOR) and the signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment with T-oligos significantly prolonged the survival time of mice inoculated intracranially with malignant glioma cells compared with that of untreated mice and those treated with control oligonucleotides (P=0.0065 and P=0.043, respectively). These results indicate that T-oligos stimulate the induction of nonapoptotic autophagic also known as type II programmed cell death and are thus promising in the treatment of malignant glioma.

Eupalmerin acetate, a novel anticancer agent from Caribbean gorgonian octocorals, induces apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway.

The marine ecosystem is a vast but largely untapped resource for potential naturally based medicines. We tested 15 compounds derived from organisms found in the Caribbean Sea (14 gorgonian octocoral-derived compounds and one sponge-derived compound) for their anticancer effects on human malignant glioma U87-MG and U373-MG cells. Eupalmerin acetate (EPA) was chosen as the lead compound based on its longer-term stability and greater cytotoxicity than those of the other compounds we tested in these cell types. EPA induced G(2)-M cell cycle arrest and apoptosis via the mitochondrial pathway; it translocated Bax from the cytoplasm to the mitochondria and dissipated the mitochondrial transmembrane potential in both cell types. EPA was found to increase phosphorylated c-Jun NH(2)-terminal kinase (JNK) by >50% in both U87-MG and U373-MG cells. A specific JNK inhibitor, SP600125, inhibited EPA-induced apoptosis, confirming the involvement of the JNK pathway in EPA-induced apoptotic cell death. Furthermore, 7 days of daily intratumoral injections of EPA significantly suppressed the growth of s.c. malignant glioma xenografts (P < 0.01, on day 19). These results indicate that EPA is therapeutically effective against malignant glioma cells in vitro and in vivo and that it, or a similar marine-based compound, may hold promise as a clinical anticancer agent.

Autophagic cell death of malignant glioma cells induced by a conditionally replicating adenovirus.

BACKGROUND: Conditionally replicating adenoviruses (CRAds) can be engineered to replicate selectively in cancer cells and cause cancer-specific cell lysis; thus they are considered a promising cancer therapy. METHODS: To elucidate the mechanisms by which CRAds induce cancer-specific cell death, we infected normal human fibroblasts (MRC5, telomerase negative), human malignant glioma (U373-MG and U87-MG), human cervical cancer (HeLa), and human prostate cancer (PC3) cells (all telomerase positive) with CRAds regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad) or control nonreplicating adenoviruses (Ad-GFP). Nonapoptotic autophagy was assessed in Ad-GFP- and hTERT-Ad-infected cells by examining cell morphology, the development of acidic vesicular organelles, and the conversion of microtubule-associated protein 1 light chain 3 from the cytoplasmic form to the autophagosome membrane form; signaling via mammalian target of rapamycin (mTOR), an autophagy-associated molecule, was monitored by western blot analysis. We also compared the growth of subcutaneous gliomas in nude mice that were treated by intratumoral injection with Ad-GFP or hTERT-Ad. Survival of athymic mice carrying intracranial gliomas treated by intratumoral injection with Ad-GFP or hTERT-Ad was compared by using the Kaplan-Meier method and the Cox-Mantel log-rank analysis. All statistical tests were two-sided. RESULTS: hTERT-Ad induced tumor-specific autophagic cell death in tumor cells and in subcutaneous gliomas. hTERT-Ad-induced autophagy was associated with hTERT-Ad infection kinetics. The mTOR signaling pathway was suppressed in tumor cells and in subcutaneous gliomas treated with hTERT-Ad compared with GFP-Ad or no treatment as shown by reduced phosphorylation of mTOR's downstream target p70S6 kinase (p70S6K). hTERT-Ad treatment of mice (n = 7) slowed growth of subcutaneous gliomas (mean tumor volume = 39 mm3, 95% confidence interval [CI] = 23 to 54 mm3) compared with GFP-Ad treatment (n = 7) (mean tumor volume = 200 mm3, 95% CI = 149 to 251 mm3) at day 7 (volume difference = 161 mm3, 95% CI = 126 to 197 mm3; P < .001). Mice carrying intracranial tumors that were treated with three intratumoral injections of hTERT-Ad survived longer (53 days) than after treatment with GFP-Ad (29 days) (seven mice per group, difference = 24 days, 95% CI = 20 to 28 days; P < .001). CONCLUSIONS: hTERT-Ad may kill telomerase-positive cancer cells by inducing autophagic cell death.