Aloe-Emodin Overcomes Anti-Cancer Drug Resistance to Temozolomide and Prevents Colony Formation and Migration in Primary Human Glioblastoma Cell Lines NULU and ZAR.

Glioblastoma, the most dangerous and aggressive type of CNS tumor, appears resistant to many chemotherapy drugs. In the patient-derived glioma cell lines NULU and ZAR, which exhibit drug-resistant phenotypes, we investigated the effect of combined AE (Aloe-emodin) and TMZ (temozolomide) and found a significant additive inhibitory effect on cell growth and a promising cytotoxic effect on both cell lines compared to treatment with single agents. We also examined the effect of combined AE and TMZ treatment on the drug-resistance protein MGMT. The results suggest that using AE combined with traditional drugs restores drug resistance in both primary resistant cell lines (NULU and ZAR). Furthermore, migration assays and scratch tests showed that the combined use of AE and TMZ can slow down the colony formation and migration of glioblastoma cells. These convincing results suggest that AE could be a natural adjuvant agent to potentiate the effects of traditional drugs (TMZ) and overcome drug resistance in glioblastoma cells.

Isoginkgetin-A Natural Compound to Control U87MG Glioblastoma Cell Growth and Migration Activating Apoptosis and Autophagy.

Isoginkgetin (Iso) is a natural bioflavonoid isolated from the leaves of Ginkgo biloba, this natural substance exhibits many healing properties, among which the antitumor effect stands out. Here we tested the effect of Iso on the growth of U87MG glioblastoma cells. Growth curves and MTT toxicity assays showed time and dose-dependent growth inhibition of U87MG after treatment with Iso (15/25 µM) for 1, 2, and 3 days. The cell growth block of U87MG was further investigated with the colony formation test, which showed that iso treatment for 24 h reduced colony formation. The present study also aimed to evaluate the effect of Iso on U87MG glioblastoma cell migration. The FACS analysis, on the other hand, showed that treatment with Iso 15 µM determines a blockage of the cell cycle in the S1 phase. Further investigation shows that Iso treatment of U87MG altered the protein pathways of homeostasis including autophagy and apoptosis. The present study demonstrated, for the first time, that Iso could represent an excellent adjuvant drug for the treatment of glioblastoma by simultaneously activating multiple mechanisms that control the growth and migration of neoplastic cells.

Characterization of primary glioma cell lines derived from the patients according to 2016 CNS tumour WHO classification and comparison with their parental tumours.

BACKGROUND: Gliomas represent about 80% of primary brain tumours and about 30% of malignant ones, which today don't have a resolution therapy because of their variability. A valid model for the study of new personalized therapies can be represented by primary cultures from patient's tumour biopsies. METHODS: In this study we consider 12 novel cell lines established from patients' gliomas and immunohistochemically and molecularly characterized according to the newly updated 2016 CNS Tumour WHO classification. RESULTS: Eight of these lines were glioblastoma cells, two grade III glioma cells (anaplastic astrocytoma and oligo astrocytoma) and two low grade glioma cells (grade II astrocytoma and oligodendroglioma). All cell lines were analysed by immunohistochemistry for specific glioma markers, respectively VIMENTIN, GFAP, IDH1R132, and ATRX. The methylation status of the MGMT gene promoter was also determined in all lines. The comparison of the immunohistochemical characteristics and of the MGMT methylation status of the lines with the tissues of origin shows that the cells in culture maintain the same characteristics. CONCLUSIONS: Human cancer cell lines represent a support in the knowledge of tumour biology and in drug discovery through its facile experimental manipulation. TRIAL REGISTRATION: NCT04180046.

Implication of Lactucopicrin in Autophagy, Cell Cycle Arrest and Oxidative Stress to Inhibit U87Mg Glioblastoma Cell Growth.

In this study, we propose lactucopicrin (LCTP), a natural sesquiterpene lactone from Lactucavirosa, as a molecule able to control the growth of glioblastoma continuous cell line U87Mg. The IC50 of U87Mg against LCTP revealed a strong cytotoxic effect. Daily administration of LCTP showed a dose and time-dependent reduction of GBM cell growth and viability, also confirmed by inhibition of clonogenic potential and mobility of U87Mg cells. LCTP activated autophagy in U87Mg cells and decreased the phosphorylation of proliferative signals pAKT and pERK. LCTP also induced the cell cycle arrest in G2/M phase, confirmed by decrease of CDK2 protein and increase of p53 and p21. LCTP stimulated apoptosis as evidenced by reduction of procaspase 6 and the increase of the cleaved/full-length PARP ratio. The pre-treatment of U87Mg cells with ROS scavenger N-acetylcysteine (NAC), which reversed its cytotoxic effect, showed the involvement of LCTP in oxidative stress. Finally, LCTP strongly enhanced the sensitivity of U87Mg cells to canonical therapy Temozolomide (TMZ) and synergized with this drug. Altogether, the growth inhibition of U87Mg GBM cells induced by LCTP is the result of several synergic mechanisms, which makes LCTP a promising adjuvant therapy for this complex pathology.

Effects of aloe emodin on U87MG glioblastoma cell growth: In vitro and in vivo study.

Glioblastoma, the most aggressive and malignant form of glioma, appears to be resistant to various chemotherapeutic agents. Hence other approaches have been investigated to target more pathways involved in glioblastoma development and progression. Here we investigate the anticancer effect of Aloe-Emodin (AE), an anthraquinone compound presents in the leaves of Aloe arborescens, on human glioblastoma cell line U87MG. U87MG were treated with various concentrations of AE (20 and 40 µM) for different times (24, 48, and 72 hr). Cell growth was monitored by daily cell count after treatments. Growth analysis showed that AE significantly decrease proliferation of U87MG in a time and dose dependent manner. FACS analysis demonstrates a block of cell cycle in S and G2/M phase. AE probably induced also apoptosis by releasing of apoptosis-inducing factor: PARP and Lamin activation leading to nuclear shrinkage. In addition, exposure of U87MG to AE reduced pAKT phosphorylation. AE inhibition of U87MG growth is a result of more mechanism together. Here we report that AE has a specific growth inhibition on U87MG also in in vivo. The growth of U87MG, subcutaneously injected in nude mice with severe combined immunodeficiency, is inhibited without any appreciable toxic effects on the animals after AE treatment. AE might represent a conceptually new lead antitumor adjuvant drug.

Effects of hispolon on glioblastoma cell growth.

Hispolon is a polyphenolic compound isolated from Phellinus linteus which exhibits antitumor activity. Here, we explored the effects of hispolon on human glioblastoma cells U87MG. Cell viability was examined by MTT assay. Growth was investigated by incubating cells with various concentrations of hispolon (25 and 50 µM) for 24, 48 or 72 h and daily cell count. Cell cycle and apoptosis assay were assessed by flow cytometry. Hispolon decreased cell viability in a dose- and time-dependent manner. The cell cycle distribution showed that hispolon enhanced the accumulation of the cells in G2/M phase. Hispolon decreased the expression of G1-S transition-related protein cyclin D4 but increased the expression of CDK inhibitor p21. Additionally, hispolon enhanced the expression of p53. Moreover, hispolon treatment was effective on U87MG cells in inhibiting cell viability and inducing cell apoptosis. Our results indicate that hispolon inhibits the cell viability, induces G2/M cell cycle arrest and apoptosis in glioblastoma U87MG cells, and p53 should play a role in hispolon-mediated antitumor activity.

Inhibiting effect of p-Coumaric acid on U87MG human glioblastoma cell growth.

p-Coumaric acid (pCA) is a hydroxycinnamic acid derivative commonly found in many natural products that has been extensively studied for its anticancer activity in multiple cell lines. In this report we investigated the effects of this phytochemical as adjuvant therapy to treat glioblastoma, an infaust brain tumour characterized by the acquired or innate resistance to the conventional chemotherapy temozolomide (TMZ). U87Mg glioblastoma cell growth and viability was assessed by growth rate curves and MTT assay incubating cells with 0.5 and 1 mM pCA for 24 h, 48 h and 72 h. Cell cycle analysis, performed by flow cytometry, showed that pCA led the accumulation of GBM cells in G2/M phase. Western blot analysis shows that pCA induced CDK4 cyclin-dependent kinase reduction and p53 increase, followed by induction of the CDK inhibitor p21. Furthermore, pCA treatment mediated the activation of apoptosis and the inhibition of migration of U87Mg cells. Finally, the treatment of glioblastoma cells in vitro with pCA concomitantly with the TMZ revealed a synergistic effect between the natural substance and the chemotherapy. In conclusion, our results demonstrated that pCA acts influencing the cell viability and cell cycle of U87Mg cells by promoting cell cycle arrest in G2/M phase and apoptosis.

In vitro and in vivo effect of human lactoferrin on glioblastoma growth.

OBJECT: Human lactoferrin (HLF) is a natural protein with antitumor activity. The aim of this study was to investigate the effects of HLF alone and in combination with temozolomide (TMZ), a conventional chemotherapeutic, on human glioblastoma (GBM) cells. METHODS: The authors cultured fresh human primary cell lines NMD and FN and the continuous cell line U87MG to evaluate proliferation in the presence of HLF alone at different doses (1, 10, and 100 mg/ml, and 1 mg/ml) and in combination with TMZ. In in vivo experiments they assessed tumor size reduction in CD1 nude mice carrying an orthotopic GBM xenograft and orally treated with HLF. RESULTS: Lactoferrin causes growth inhibition in the NMD and FN primary cell lines and in the U87MG continuous cell line. This inhibition seemed to be modulated by the downregulation of cyclin D1 and D4. Western blot and fluorescence-activated cell sorting analysis showed inhibition of the cell cycle in G0/G1 and G2 phases. When administered in nude mice, HLF (60 mg/kg/day) decreased tumor size about 30%, as shown in both histological analyses and high-field brain MRI. Administration of HLF with TMZ enhanced the effect of chemotherapy both in vitro and in vivo. CONCLUSIONS: This study demonstrated that HLF can inhibit GBM cell growth, suggesting that this nontoxic substance may have a role in potentiating the effect of current TMZ treatment of GBM.