Antitumor Effects of 5-Aminolevulinic Acid on Human Malignant Glioblastoma Cells.
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ABSTRACT:
SUBMITTER: Jalili-Nik M
PROVIDER: S-EPMC8199444 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Neurofibromatosis type 1 (NF1) is one of the most common neurocutaneous disorders. Some NF1 patients develop benign large plexiform neurofibroma(s) at birth, which can then transform into a malignant peripheral nerve sheath tumor (MPNST). There is no curative treatment for this rapidly progressive and easily metastatic neurofibrosarcoma. Photodynamic therapy (PDT) has been developed as an anti-cancer treatment, and 5-aminolevulinic (ALA) mediated PDT (ALA-PDT) has been used to treat cutaneous skin and oral neoplasms. Doxycycline, a tetracycline derivative, can substantially reduce the tumor burden in human and animal models, in addition to its antimicrobial effects. The purpose of this study was to evaluate the effect and to investigate the mechanism of action of combined doxycycline and ALA-PDT treatment of MPNST cells. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the combination of ALA-PDT and doxycycline significantly reduce MPNST survival rate, compared to cells treated with each therapy alone. Isobologram analysis showed that the combined treatment had a synergistic effect. The increased cytotoxic activity could be seen by an increase in cellular protoporphyrin IX (PpIX) accumulation. Furthermore, we found that the higher retention of PpIX was mainly due to increasing ALA uptake, rather than activity changes of the enzymes porphobilinogen deaminase and ferrochelatase. The combined treatment inhibited tumor growth in different tumor cell lines, but not in normal human Schwann cells or fibroblasts. Similarly, a synergistic interaction was also found in cells treated with ALA-PDT combined with minocycline, but not tetracycline. In summary, doxycycline can potentiate the effect of ALA-PDT to kill tumor cells. This increased potency allows for a dose reduction of doxycycline and photodynamic radiation, reducing the occurrence of toxic side effects in vivo.
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Project description:5-Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has the potential to kill cancer cells via apoptotic or necrotic signals that are dependent on the generation of intracellular reactive oxygen species (ROS). Celecoxib is an anti-inflammatory drug that induces intracellular ROS generation. We investigated whether the combined application of celecoxib and ALA-PDT improved the efficacy of PDT in human cholangiocarcinoma cells and in tumor bearing mice. In vitro, combined treatment of celecoxib and ALA-PDT increased phototoxicity and intracellular ROS levels after irradiation with 0.75 J/cm(2) when compared to ALA-PDT alone. Even though ROS levels increased with 0.25 J/cm(2) of irradiation, it did not influence phototoxicity. When heme oxygenase-1, a defensive protein induced by oxidative stress, was inhibited in the combined treatment group, phototoxicity was increased at both 0.25 J/cm(2) and 0.75 J/cm(2) of irradiation. We identified the combined effect of ALA-PDT and celecoxib through the increase of oxidative stress such as ROS. In vivo, about 40% tumor growth inhibition was observed with combined application of ALA-PDT and celecoxib when compared to ALA-PDT alone. The combined application of ALA-PDT and celecoxib could be an effective therapy for human cholangiocarcinoma. Moreover, use of a heme oxygenase-1 inhibitor with PDT could play an important role for management of various tumors involving oxidative stress.
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Project description:ObjectiveGlioblastoma multiforme (GBM) is the deadliest type of primary brain tumors, and the survival of patients is estimated to be only about one year. This study, for the first time, investigated the cytotoxic effects of auraptene on U87 GBM cell line.Materials and methodsThe cellular toxicity was measured by the MTT assay following 24 and 48-hr treatment with different concentrations of auraptene (0-400μg/ml). Apoptosis was evaluated by sub-G1 peak in cell cycle analysis of propidium-iodide- stained nuclei. Moreover, to determine the Bax, Bcl-2, MCP-1, NF-κB, IL-1β, and p53 genes expression, we used real-time polymerase chain reaction (RT-PCR).ResultsThe results revealed that auraptene reduced the viability of U87 cells concentration- and time-dependently with IC50 values of 108.9 and 79.17μg/ml obtained for 24 and 48-hr treatments, respectively. Also, sub-G1 population was significantly increased following 24 (p<0.05 and p<0.001) and 48 (p<0.001) hours of treatment. The quantitative real-time RT-PCR showed an up-regulation in Bax, NF-κB, IL-1β, and p53 but a down-regulation in MCP-1 and Bcl-2 genes expression.ConclusionThis study showed that auraptene triggered apoptosis probably through Bax/Bcl-2 regulation, blocked cell cycle progression and inhibited proliferation in U87 GBM cells. Taken together, auraptene can be utilized as an effective natural medicine against GBM, after complementary studies.
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Project description:Over recent years, many authors discussed the effects of different natural compounds on glioblastoma (GBM). Due to its capacity to impair survival and progression of different cancer types, saffron extract (SE), named crocetin (CCT), is particularly noteworthy. In this work, we elucidated the antitumor properties of crocetin in glioma in vivo and in vitro models for the first time. The in vitro results showed that the four tumor cell lines observed in this study (U251, U87, U138, and U373), which were treated with increasing doses of crocetin, showed antiproliferative and pro-differentiative effects as demonstrated by a significant reduction in the number of viable cells, deep changes in cell morphology, and the modulation of mesenchymal and neuronal markers. Indeed, crocetin decreased the expression of Cluster of Differentiation CD44, CD90, CXCR4, and OCT3/4 mesenchymal markers, but increased the expression of ?III-Tubulin and neurofilaments (NFH) neuronal linage-related markers. Epigenetic mechanisms may modulate these changes, since Histone Deacetylase, HDAC1 and HDAC3 were downmodulated in U251 and U87 cells, whereas HDAC1 expression was downmodulated in U138 and U373 cells. Western blotting analyses of Fatty Acid Synthase, FASN, and CD44 resulted in effective inhibition of these markers after CCT treatment, which was associated with important activation of the apoptosis program and reduced glioma cell movement and wound repair. The in vivo studies aligned with the results obtained in vitro. Indeed, crocetin was demonstrated to inhibit the growth of U251 and U87 cells that were subcutaneously injected into animal models. In particular, the Tumor To Progression or TTP values and Kaplan-Meier curves indicated that crocetin had more major effects than radiotherapy alone, but similar effects to temozolomide (TMZ). An intra-brain cell inoculation of a small number of luciferase-transfected U251 cells provided a model that was able to recapitulate recurrence after surgical tumor removal. The results obtained from the orthotopic intra-brain model indicated that CCT treatment increased the disease-free survival (DFS) and overall survival (OS) rates, inducing a delay in appearance of a detectable bioluminescent lesion. CCT showed greater efficacy than Radio Therapy (RT) but comparable efficacy to temozolomide in xenograft models. Therefore, we aimed to continue the study of crocetin's effects in glioma disease, focusing our attention on the radiosensitizing properties of the natural compound and highlighting the ways in which this was realized.
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Project description:Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) has become the main treatment modality in malignant gliomas. However unlike glioblastomas, there are inconsistent result about fluorescence status in WHO grade III gliomas. Here, we show that mutational status of IDH1 is linked to 5-ALA fluorescence. Using genetically engineered malignant glioma cells harboring wild type (U87MG-IDH1WT) or mutant (U87MG-IDH1R132H) IDH1, we demonstrated a lag in 5-ALA metabolism and accumulation of protoporphyrin IX (PpIX) in U87MG-IDH1R132Hcells. Next, we used liquid chromatography-mass spectrometry (LC-MS) to screen for tricarboxylic acid (TCA) cycle-related metabolite changes caused by 5-ALA exposure. We observed low baseline levels of NADPH, an essential cofactor for the rate-limiting step of heme degradation, in U87MG-IDH1R132H cells. High levels of NADPH are required to metabolize excessive 5-ALA, giving a plausible reason for the temporarily enhanced 5-ALA fluorescence in mutant IDH1 cells. This hypothesis was supported by the results of metabolic screening in human malignant glioma samples. In conclusion, we have discovered a relationship between enhanced 5-ALA fluorescence and IDH1 mutations in WHO grade III gliomas. Low levels of NADPH in tumors with mutated IDH1 is responsible for the enhanced fluorescence.
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Project description:Glioblastoma is the leading malignant glioma with a poor prognosis. This study aimed to investigate the antitumor effects of natural killer cells in combination with temozolomide as the standard chemotherapeutic agent for glioblastoma. Using a simple, feeder-less, and chemically defined culture method, we expanded human peripheral blood mononuclear cells and assessed the receptor expression, natural killer cell activity, and regulatory T cell frequency in expanded cells. Next, using the standard human glioblastoma cell lines (temozolomide-sensitive U87MG, temozolomide-resistant T98G, and LN-18), we assessed the ligand expressions of receptors on natural killer cells. Furthermore, the antitumor effects of the combination of the expanded natural killer cells and temozolomide were assessed using growth inhibition assays, apoptosis detection assays, and senescence-associated ?-galactosidase activity assays in the glioblastoma cell lines. Novel culture systems were sufficient to attain highly purified (>98%), expanded (>440-fold) CD3-/CD56+ peripheral blood-derived natural killer cells. We designated the expanded population as genuine induced natural killer cells. Genuine induced natural killer cells exhibited a high natural killer activity and low regulatory T cell frequency compared with lymphokine-activated killer cells. Growth inhibition assays revealed that genuine induced natural killer cells inhibited the glioblastoma cell line growth but enhanced temozolomide-induced inhibition effects in U87MG. Apoptosis detection assays revealed that genuine induced natural killer cells induced apoptosis in the glioblastoma cell lines. Furthermore, senescence-associated ?-galactosidase activity assays revealed that temozolomide induced senescence in U87MG. Genuine induced natural killer cells induce apoptosis in temozolomide-sensitive and temozolomide-resistant glioblastoma cells and enhances temozolomide-induced antitumor effects in different mechanisms. Hence, the combination of genuine induced natural killer cells and temozolomide may prove to be a promising immunochemotherapeutic approach in patients with glioblastoma if the antitumor effects in vivo can be demonstrated.
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Project description:Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of RalB also inhibits cell growth in temozolomide-resistant GBM cells. Overall, our work shows that GBM cells are especially sensitive to Ral-GTPase availability. Therefore, we propose that the inactivation of Ral-GTPases may be a reliable therapeutic approach to prevent GBM progression and recurrence.
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