Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.     

Detoxication of Benzo[a]pyrene-7,8-dione by Sulfotransferases (SULTs) in Human Lung Cells

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Human aldo-keto reductases catalyze the metabolic activation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active o-quinones. Benzo[a]pyrene-7,8-dione a representative PAH o-quinone is reduced back to the corresponding catechol to generate a futile redox-cycle. We investigated whether sulfonation of PAH catechols by human sulfotransferases (SULT) could intercept the catechol in human lung cells. RT-PCR identified SULT1A1, -1A3, and -1E1 as the isozymes expressed in four human lung cell lines. The corresponding recombinant SULTs were examined for their substrate specificity. Benzo[a]pyrene-7,8-dione was reduced to benzo[a]pyrene-7,8-catechol by dithiothreitol under anaerobic conditions and then further sulfonated by the SULTs in the presence of 3'-[(35)S]phosphoadenosine 5'-phosphosulfate as the sulfonate group donor. The human SULTs catalyzed the sulfonation of benzo[a]pyrene-7,8-catechol and generated two isomeric benzo[a]pyrene-7,8-catechol O-monosulfate products that were identified by reversed phase HPLC and by LC-MS/MS. The various SULT isoforms produced the two isomers in different proportions. Two-dimensional (1)H and (13)C NMR assigned the two regioisomers of benzo[a]pyrene-7,8-catechol monosulfate as 8-hydroxy-benzo[a]pyrene-7-O-sulfate (M1) and 7-hydroxy-benzo[a]pyrene-8-O-sulfate (M2), respectively. The kinetic profiles of three SULTs were different. SULT1A1 gave the highest catalytic efficiency (k(cat)/K(m)) and yielded a single isomeric product corresponding to M1. By contrast, SULT1E1 showed distinct substrate inhibition and formed both M1 and M2. Based on expression levels, catalytic efficiency, and the fact that the lung cells only produce M1, it is concluded that the major isoform that can intercept benzo[a]pyrene-7,8-catechol is SULT1A1.     

Metabolic patterns and biotransformation activities of resveratrol in human glioblastoma cells: relevance with therapeutic efficacies

Background

Trans-resveratrol rather than its biotransformed monosulfate metabolite exerts anti-medulloblastoma effects by suppressing STAT3 activation. Nevertheless, its effects on human glioblastoma cells are variable due to certain unknown reason(s).

Methodology/Principal Findings

Citing resveratrol-sensitive UW228-3 medulloblastoma cell line and primarily cultured rat brain cells/PBCs as controls, the effect of resveratrol on LN-18 human glioblastoma cells and its relevance with metabolic pattern(s), brain-associated sulfotransferase/SULT expression and the statuses of STAT3 signaling and protein inhibitor of activated STAT3 (PIAS3) were elucidated by multiple experimental approaches. Meanwhile, the expression patterns of three SULTs (SULT1A1, 1C2 and 4A1) in human glioblastoma tumors were profiled immunohistochemically. The results revealed that 100 µM resveratrol-treated LN-18 generated the same metabolites as UW228-3 cells, while additional metabolite in molecular weight of 403.0992 in negative ion mode was found in PBCs. Neither growth arrest nor apoptosis was found in resveratrol-treated LN-18 and PBC cells. Upon resveratrol treatment, the levels of SULT1A1, 1C2 and 4A1 expression in LN-18 cells were more up-regulated than that expressed in UW228-3 cells and close to the levels in PBCs. Immunohistochemical staining showed that 42.0%, 27.1% and 19.6% of 149 glioblastoma cases produced similar SULT1A1, 1C2 and 4A1 levels as that of tumor-surrounding tissues. Unlike the situation in UW228-3 cells, STAT3 signaling remained activated and its protein inhibitor PIAS3 was restricted in the cytosol of resveratrol-treated LN-18 cells. No nuclear translocation of STAT3 and PIAS3 was observed in resveratrol-treated PBCs. Treatment with STAT3 chemical inhibitor, AG490, committed majority of LN-18 and UW228-3 cells but not PBCs to apoptosis within 48 hours.

Conclusions/Significance

LN-18 glioblastoma cells are insensitive to resveratrol due to the more inducible brain-associated SULT expression, insufficiency of resveratrol to suppress activated STAT3 signaling and the lack of PIAS3 nuclear translocation. The findings from PBCs suggest that an effective anticancer dose of resveratrol exerts little side effect on normal brain cells.     

Sulfotransferase 2A1 forms estradiol-17-sulfate and celecoxib switches the dominant product from estradiol-3-sulfate to estradiol-17-sulfate

Using recombinant sulfotransferases (SULTs) expressed in E. coli, β-estradiol (E2) sulfonation was examined to determine which SULT enzyme is responsible for producing E2-17-sulfate (E2-17-S). SULTs 1A1*1, 1A1*2, 1A3, 1E1 and 2A1 all sulfated E2 to varying extents. No activity was observed with SULT1B1. Among the SULTs studied, SULT2A1 produced primarily E2-3-sulfate (E2-3-S), but also some E2-17-S and trace amounts of E2 disulfate. SULT2A1 had a K_m value of 1.52 μM for formation of E2-3-S and 2.95 μM for formation of E2-17-S. SULT2A1 had the highest V_max of 493 pmol/min/mg protein for formation of E2-3-S, which was 8.8- and 47-fold higher than the maximal rates of formation of E2-17-S and E2 disulfate, respectively. SULT2A1 formed E2-3-S more efficiently. However, when celecoxib (0–160 μM) was included in the incubation with either SULT2A1 or human liver cytosol, sulfonation switched from E2-3-S to E2-17-S in a concentration-dependent manner. The ratio of E2-17-S/E2-3-S went up to 15 with SULT2A1, and was saturated at 1 with human liver cytosol. In both cases, more E2-17-S was formed, with the unreacted E2 remained unchanged, suggesting celecoxib probably bound to a separate effector site to cause a conformational change in SULT2A1, which favored production of E2-17-S. The ability of celecoxib to alter the position of sulfonation of E2 may in part explain its success in the experimental prevention and treatment of breast cancer.     

Crystal structure of mSULT1D1, a mouse catecholamine sulfotransferase

In mammals, sulfonation as mediated by specific cytosolic sulfotransferases (SULTs) plays an important role in the homeostasis of dopamine and other catecholamines. To gain insight into the structural basis for dopamine recognition/binding, we determined the crystal structure of a mouse dopamine-sulfating SULT, mouse SULT1D1 (mSULT1D1). Data obtained indicated that mSULT1D1 comprises of a single α/β domain with a five-stranded parallel β-sheet. In contrast to the structure of the human SULT1A3 (hSULT1A3)-dopamine complex previously reported, molecular modeling and mutational analysis revealed that a water molecule plays a critical role in the recognition of the amine group of dopamine by mSULT1D1. These results imply differences in substrate binding between dopamine-sulfating SULTs from different species.     

Molecular prognostic of nine parthanatos death-related genes in glioma,particularly in COL8A1 identification

Post-operative progression and chemotherapy resistance are the main causes of treatment failure in glioma patients. There is a lack of ideal prediction models for post-operative glioma patient progression and drug sensitivity. We aimed to develop a prognostic model of parthanatos mRNA biomarkers for glioma outcomes. A total of 11 parthanatos genes were obtained from ParthanatosCluster database. ConsensusClusterPlus and R “Limma” package were used to cluster The Cancer Genome Atlas (TCGA)-glioma cohort and analyze the differential mRNAs. Univariate Cox regression analysis, random survival forest model, and least absolute shrinkage and selection operator (LASSO) regression analysis were used to determine the nine ParthanatosScore prognostic genes combination. ParthanatosScore was verified by 656 patients and 979 patients in TCGA and CGCA-LGG/GBM datasets. Differences in genomic mutations, tumor microenvironments, and functional pathways were assessed. Drug response prediction was performed using pRRophetic. Kaplan–Meier survival analysis was analyzed. Finally, COL8A1 was selected to evaluate its potential biological function and drug sensitivity of temozolomide and AZD3759 in glioma cells. ParthanatosScore obtained a combination of nine glioma prognostic genes, including CD58, H19, TNFAIP6, FTLP3, TNFRSF11B, SFRP2, LOXL1, COL8A1, and FABP5P7. In the TCGA-LGG/GBM dataset, glioma prognosis was poor in high ParthanatosScore. Low-score glioma patients were sensitive to AZD3759_1915, AZD5582_1617, AZD8186_1918, Dasatinib_1079, and Temozolomide_1375, while high-score patients were less sensitive to these drugs. Compared with HA cells, COL8A1 was significantly over-expressed in LN229 and U251 cells. Silencing COL8A1 inhibited the malignant characterization of LN229 and U251 cells. Temozolomide and AZD3759 also promoted parthanatos gene expression in glioma cells. Temozolomide and AZD3759 inhibited COL8A1 expression and cell viability and promoted apoptosis in glioma cells and PGM cells. ParthanatosScore can accurately predict clinical prognosis and drug sensitivity after glioma surgery. Silencing COL8A1 inhibited the malignant characterization. Temozolomide and AZD3759 inhibited COL8A1 expression and cell viability and promoted apoptosis and parthanatos gene expression, which is a target to improve glioma.     

Glioblastoma cell secretome: analysis of three glioblastoma cell lines reveal 148 non-redundant proteins

Glioblastoma multiforme (GBM) is the most aggressive among human gliomas with poor prognosis. Study of tumor cell secretome - proteins secreted by cancer cell lines, is a powerful approach to discover potential diagnostic or prognostic biomarkers. Here we report, for the first time, proteins secreted by three GBM cell lines, HNGC2, LN229 and U87MG. Analysis of the conditioned media of these cell lines by LC-MS/MS using ESI-IT mass spectrometer (LTQ) resulted in the confident identification of 102, 119 and 64 proteins, respectively. Integration of the results from all the three cell lines lead to a dataset of 148 non-redundant proteins. Subcellular classification using Genome Ontology indicated that 42% of the proteins identified belonged to extracellular or membrane proteins, viz. Vinculin, Tenascin XB, SERPIN F1 and TIMP-1. 52 proteins matched with the secretomes of 11 major cancer types reported earlier whereas remaining 96 are unique to our study. 25 protein identifications from the dataset represent proteins related to GBM or other cancer tissues as per Human Protein Atlas; at least 22 are detectable in plasma, 11 of them being reported even in cerebrospinal fluid. Our study thus provides a valuable resource of GBM cell secretome with potential for further investigation as GBM biomarkers.     

Saponin 1 Induces Apoptosis and Suppresses NF-κB-Mediated Survival Signaling in Glioblastoma Multiforme (GBM)

Saponin 1 is a triterpeniod saponin extracted from Anemone taipaiensis, a traditional Chinese medicine against rheumatism and phlebitis. It has also been shown to exhibit significant anti-tumor activity against human leukemia (HL-60 cells) and human hepatocellular carcinoma (Hep-G2 cells). Herein we investigated the effect of saponin 1 in human glioblastoma multiforme (GBM) U251MG and U87MG cells. Saponin 1 induced significant growth inhibition in both glioblastoma cell lines, with a 50% inhibitory concentration at 24 h of 7.4 µg/ml in U251MG cells and 8.6 µg/ml in U87MG cells, respectively. Nuclear fluorescent staining and electron microscopy showed that saponin 1 caused characteristic apoptotic morphological changes in the GBM cell lines. Saponin 1-induced apoptosis was also verified by DNA ladder electrophoresis and flow cytometry. Additionally, immunocytochemistry and western blotting analyses revealed a time-dependent decrease in the expression and nuclear location of NF-κB following saponin 1 treatment. Western blotting data indicated a significant decreased expression of inhibitors of apoptosis (IAP) family members,(e.g., survivin and XIAP) by saponin 1. Moreover, saponin 1 caused a decrease in the Bcl-2/Bax ratio and initiated apoptosis by activating caspase-9 and caspase-3 in the GBM cell lines. These findings indicate that saponin 1 inhibits cell growth of GBM cells at least partially by inducing apoptosis and inhibiting survival signaling mediated by NF-κB. In addition, in vivo study also demonstrated an obvious inhibition of saponin 1 treatment on the tumor growth of U251MG and U87MG cells-produced xenograft tumors in nude mice. Given the minimal toxicities of saponin 1 in non-neoplastic astrocytes, our results suggest that saponin 1 exhibits significant in vitro and in vivo anti-tumor efficacy and merits further investigation as a potential therapeutic agent for GBM.     

A comparative study of the sulfation of bile acids and a bile alcohol by the Zebra danio (Danio rerio) and human cytosolic sulfotransferases (SULTs)

The current study was designed to examine the sulfation of bile acids and bile alcohols by the Zebra danio (Danio rerio) SULTs in comparison with human SULTs. A systematic analysis using the fifteen Zebra danio SULTs revealed that SULT3 ST2 and SULT3 ST3 were the major bile acid/alcohol-sulfating SULTs. Among the eleven human SULTs, only SULT2A1 was found to be capable of sulfating bile acids and bile alcohols. To further investigate the sulfation of bile acids and bile alcohols by the two Zebra danio SULT3 STs and the human SULT2A1, pH-dependence and kinetics of the sulfation of bile acids/alcohols were analyzed. pH-dependence experiments showed that the mechanisms underlying substrate recognition for the sulfation of lithocholic acid (a bile acid) and 5α-petromyzonol (a bile alcohol) differed between the human SULT2A1 and the Zebra danio SULT3 ST2 and ST3. Kinetic analysis indicated that both the two Zebra danio SULT3 STs preferred petromyzonol as substrate compared to bile acids. In contrast, the human SULT2A1 was more catalytically efficient toward lithocholic acid than petromyzonol. Collectively, the results imply that the Zebra danio and human SULTs have evolved to serve for the sulfation of, respectively, bile alcohols and bile acids, matching the cholanoid profile in these two vertebrate species.     

Hydroxylated serotonin and dopamine as substrates and inhibitors for human cytosolic SULT1A3

Sulfation as catalyzed by the cytosolic sulfotransferases (SULTs) is known to play an important role in the regulation and homeostasis of monoamine neurotransmitters. The current study was designed to examine the occurrence of the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine by human cytosolic SULTs and to investigate the inhibitory effects of these hydroxylated derivatives on the sulfation of their unhydroxylated counterparts, serotonin and dopamine. A systematic study using 11 known human cytosolic SULTs revealed SULT1A3 as the responsible enzyme for the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine. The pH-dependence and kinetic constants of SULT1A3 with 7-hydroxyserotonin or 6-hydroxydopamine as substrate were determined. The inhibitory effects of 7-hydroxyserotonin and 6-hydroxydopamine on the sulfation of serotonin and dopamine were evaluated. Kinetic analyses indicated that the mechanism underlying the inhibition by these hydroxylated monoamine derivatives is of a competitive-type. Metabolic labeling experiments showed the generation and release of [³⁵S]sulfated 7-hydroxyserotonin and [³⁵S]sulfated 6-hydroxydopamine when SK-N-MC human neuroblastoma cells were labeled with [³⁵S]sulfate in the presence of 7-hydroxyserotonin or 6-hydroxydopamine. Upon transfection of the cells with siRNAs targeted at SULT1A3, diminishment of the SULT1A3 protein and concomitantly the sulfating activity toward these hydroxylated monoamines was observed. Taken together, these results indicated clearly the involvement of sulfation in the metabolism of 7-hydroxyserotonin and 6-hydroxydopamine. By serving as substrates for SULT1A3, these hydroxylated monoamines may interfere with the homeostasis of endogenous serotonin and dopamine.     

Lys05 induces lysosomal membrane permeabilization and increases radiosensitivity in glioblastoma

Glioblastoma (GBM) is one of the most malignant primary brain tumors and its prognosis is very poor. Lysosome-dependent cell death is mainly caused by lysosomal membrane permeabilization (LMP), a process in which the lysosome loses its membrane integrity and lysosomal contents are released into the cytosol. Lysosomotropic agent, a kind of compound that selectively accumulates in the lysosomes, is one of the most important inducers of LMP. As a newly-synthetic lysosomotropic agent, Lys05 showed efficient autophagy inhibiting and antitumor effect. But its mechanisms are not well illustrated. Here, we studied whether Lys05 has antiglioma activity. We found that Lys05 decreased cell viability and reduced cell growth of glioma U251 and LN229 cells. After Lys05 treatment, autophagic flux is inhibited and lysosome function is impaired. We also found that Lys05 caused LMP and mitochondrial depolarization. Finally, Lys05 increased radiosensitivity in an LMP-dependent manner. For the first time, our findings indicate that LMP contributes to radiosensitivity in GBM cells. Therefore, LMP inducer, Lys05 might be a promising compound in the treatment of GBM cells.     

Sulfation of ractopamine and salbutamol by the human cytosolic sulfotransferases

Feed additives such as ractopamine and salbutamol are pharmacologically active compounds, acting primarily as β-adrenergic agonists. This study was designed to investigate whether the sulfation of ractopamine and salbutamol may occur under the metabolic conditions and to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating two major feed additive compounds, ractopamine and salbutamol. A metabolic labelling study showed the generation and release of [(35)S]sulfated ractopamine and salbutamol by HepG2 human hepatoma cells labelled with [(35)S]sulfate in the presence of these two compounds. A systematic analysis using 11 purified human SULTs revealed SULT1A3 as the major SULT responsible for the sulfation of ractopamine and salbutamol. The pH dependence and kinetic parameters were analyzed. Moreover, the inhibitory effects of ractopamine and salbutamol on SULT1A3-mediated dopamine sulfation were investigated. Cytosol or S9 fractions of human lung, liver, kidney and small intestine were examined to verify the presence of ractopamine-/salbutamol-sulfating activity in vivo. Of the four human organs, the small intestine displayed the highest activity towards both compounds. Collectively, these results imply that the sulfation mediated by SULT1A3 may play an important role in the metabolism and detoxification of ractopamine and salbutamol.     

Role of Sonic hedgehog signaling in cell cycle,oxidative stress,and autophagy of temozolomide resistant glioblastoma

The first-line chemotherapy treatment for Glioblastoma (GBM) - the most aggressive and frequent brain tumor - is temozolomide (TMZ). The Sonic hedgehog (SHH) pathway is involved with GBM tumorigenesis and TMZ chemoresistance. The role of SHH pathway inhibition in the potentiation of TMZ's effects using T98G, U251, and GBM11 cell lines is investigated herein. The combination of GANT-61 and TMZ over 72 hr suggested a synergistic effect. All TMZ-resistant cell lines displayed a significant decrease in cell viability, increased DNA fragmentation and loss of membrane integrity. For T98G cells, G₂/M arrest was observed, while U251 cells presented a significant increase in reactive oxygen species production and catalase activity. All the cell lines presented acidic vesicles formation correlated to Beclin-1 overexpression. The combined treatment also enhanced GLI1 expression, indicating the presence of select resistant cells. The selective inhibition of the SHH pathway potentiated the cytotoxic effect of TMZ, thus becoming a promising in vitro strategy for GBM treatment.     

Swelling‐induced chloride current in glioblastoma proliferation,migration, and invasion

Glioblastoma (GBM) remains as the most common and aggressive brain tumor. The survival of GBM has been linked to the aberrant activation of swelling‐induced chloride current I_Cl,swell. In this study, we investigated the effects of I_Cl,swell on cell viability, proliferation, and migration in the human GBM cell lines, U251 and U87, using a combination of patch clamp electrophysiology, MTT, colony formation, wound healing assays and Western immunoblotting. First, we showed that the specific inhibitor of I_Cl,swell, DCPIB, potently reduced the I_Cl,swell in U87 cells. Next, in both U87 and U251 cells, we found that DCPIB reduced GBM viability, proliferation, colony formation, migration, and invasion. In addition, our Western immunoblot assay showed that DCPIB‐treated U251 cells had a reduction in JAK2, STAT3, and Akt phosphorylation, thus, suggesting that DCPIB potentially suppresses GBM functions through inhibition of the JAK2/STAT3 and PI3K/Akt signaling pathways. Therefore, the I_Cl,swell may be a potential drug target for GBM.     

Influenza A virus infection activates cholesterol sulfotransferase (SULT2B1b) in the lung of female C57BL/6 mice

Cytosolic sulfotransferases (SULTs) catalyze the sulfation of hormones, neurotransmitters, and xenobiotics, increasing their water solubility. SULTs are not only important for xenobiotic detoxification but they also play important biological roles in the regulation of the activities of various biosignaling molecules and other cellular functions. In this study, we investigated the effects of influenza A virus lung infection on the expression of SULTs in the lung, brain, and liver of female C57BL/6 mice. Our results demonstrate for the first time that SULT2B1b enzyme activity and protein expression are significantly up-regulated in the lung and brain of female mice in response to lung influenza A virus infection. Real-time quantitative PCR results are consistent with Western blot and enzymatic activity data. In mouse liver, mSULT2B1b is not significantly changed. Enzyme activities, protein expression, and mRNA expression of SULT1A1 and SULT2A1 in the lung, brain, and liver of mice were not significantly affected by the infection. The induction of SULT2B1b may be used to inactivate natural liver X receptor ligands and activate the proliferation of T cells in response to influenza A virus infection in the lung and brain of mice. Our results raise the possibility that regulation of SULT2B1b may influence acquired immune responses to infectious diseases.     

Curcumin increases efficiency of γ-irradiation in gliomas by inhibiting Hedgehog signaling pathway

It was reported that γ-irradiation had a controversial therapeutic effect on glioma cells. We aimed to investigate the cytotoxic effect on the glioma cells induced by γ-irradiation and explore the treatment to rescue the phenotype alteration of remaining cells. We used transwell assay to detect the glioma cell invasion and migration capacity. Cell proliferation and apoptosis were tested by the CCK-8 assay and flow cytometry respectively. Western Blot was used to detect the activity of Hedgehog signaling pathway and Epithelial-to-Mesenchymal Transition (EMT) status. γ-irradiation showed cytotoxic effect on LN229 cells in vitro, whereas this contribution was limited in U251 cells. However, it could significantly stimulated EMT process in both LN229 and U251. Curcumin (CCM) could rescue EMT process induced by γ-irradiation via the suppression of Gli1 and the upregulation of Sufu. The location and expression of EMT markers were also verified by Immunofluorescence. Immunohistochemistry assay was used on intracranial glioma tissues of nude mice. The capacities of cell migration and invasion were suppressed with combined therapy. This research showed Curcumin could rescue the EMT process induced by γ-irradiation via inhibiting the Hedgehog signaling pathway and potentiate the cell cytotoxic effect in vivo and in vitro.