ABSTRACT
Objectives: Cardiac glycosides are used as potential anti-cancer agents due to their effects on the inhibition of proliferation and induction of apoptosis and/or autophagy in cancer cells. Herein, we aimed to study the potential signaling pathways taken role in differential cell-death properties of AnvirzelTM which is consisted of two toxic cardiac glycosides (oleandrin and oleandrigenin), in U87 human glioblastoma cells.Methods: The anti-proliferative and anti-migratory effects of AnvirzelTM were assessed in U87 cells by WST-1 assay and wound healing assay, respectively. After treatment of AnvirzelTMwith doses of 10, 25, 50, 100 and 250 µg/ml, expression levels of proteins related to cell death were investigated by Western blot.Results: Anvirzel™ markedly inhibited the growth of U87 cells in a time- and dose-dependent manner following 24 h and 48 h treatments (p < 0.05). In addition, it was found that Anvirzel™ inhibited GSK-3, NOS and HIF1-α expressions whereas activated ERK in U87 cells compared to vehicle (p < 0.05).Discussion: The results suggested that AnvirzelTM regulated cell death distinctly from apoptosis in human glioblastoma cells. Further studies are required for validation of mechanistic insights about the potential signaling pathways taken role in differential cell death properties of AnvirzelTM.
Subject(s)
Cardenolides/pharmacology , Cell Movement/drug effects , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Cardiac Glycosides/pharmacology , Cell Death/drug effects , Cell Death/physiology , Cell Line, Tumor , Cell Movement/physiology , Cell Survival/drug effects , Cell Survival/physiology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , HumansABSTRACT
Background: There is lack of data on the effect of stereotactic radiosurgery in modulation of the immune system for cancer patients with metastatic brain tumours. Therefore, we investigated the change in levels of immunoregulatory molecules after Gamma Knife radiosurgery (GKR) and whole brain radiation therapy (WBRT) in patients with brain metastases.Methods: Peripheral blood samples were collected from 15 patients who received GKR, nine patients who received WBRT for brain metastases and 10 healthy controls. Samples were obtained at three time points such as before, 1h after and 1 week after the index procedure for patients treated with GKR or WBRT. All patients' demographic data and radiosurgical parameters were retrospectively reviewed. We analyzed the change in the levels of T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death ligand-1 (PD-L1), and cytokines such as IL-2, IL-10, IFN-γ, TNF-α after GKR and WBRT using Enzyme-linked immunosorbent assays (ELISA).Results: Baseline level of IFN-γ was found to be lower and that of PD-L1 was higher in the GKR group compared to WBRT group and healthy controls (p < 0.05 and p < 0.01, respectively). Levels of IFN-γ and IL-2 were increased (p < 0.01 and p < 0.01, respectively), while CTLA-4 and PD-L1 were decreased (p = 0.05 and p = 0.01, respectively) after GKR compared to pre-GKR levels, while there was no change after WBRT.Conclusion: GKR regulates immunoregulatory molecules towards enhancing the immune system, while WBRT did not exert any effect. These findings suggested that treatment of metastatic brain lesion with GKR might stimulate a systemic immune response against the tumour.
Subject(s)
Brain Neoplasms , Radiosurgery , Brain , Brain Neoplasms/radiotherapy , Brain Neoplasms/surgery , Humans , Immunity , Retrospective StudiesABSTRACT
BACKGROUND: Patients with brain metastasis from melanoma have a dismal prognosis with poor survival time. Gamma Knife (GK) is an effective treatment to control brain metastasis from melanoma. Thymoquinone (TQ) has emerged as a potential therapeutic option due to its antiproliferative effects on various cancers. The purpose of the study was to assess the effect of GK on B16-F10 melanoma cells in vitro and intracerebral melanoma in vivo, and its synergistic effect in combination with TQ. METHODS: The effects of GK and combination treatment of GK and TQ were studied on B16-F10 melanoma cells by evaluating cytotoxicity with an adenosine triphosphate assay, apoptosis by acridine orange staining, and genotoxicity by comet assay. Western blot analysis was performed to investigate the expression of STAT3, p-STAT3 (Tyr705), JAK2, p-JAK2, caspase-3, Bax, Bcl-2, survivin, and ß-actin. Expression of inflammatory cytokines was assessed by enzyme-linked immunosorbent assay. GK alone and in combination with TQ was assessed in an established intracerebral melanoma tumor in mice. RESULTS: The effects of GK on cytotoxicity, genotoxicity, and apoptosis were enhanced by TQ in B16-F10 melanoma cells. GK induced apoptosis through inhibition of p-STAT3 expression, which in turn regulated pro- and antiapoptotic proteins such as caspase-3, Bax, Bcl-2, and survivin. Adding TQ to GK irradiation further enhanced this apoptotic effect of GK irradiation. GK was shown to reduce the levels of tumor-related inflammatory cytokines in B16-F10 melanoma cells. This effect was more pronounced when TQ was added to GK irradiation. GK with 15 Gy increased the survival of mice with intracerebral melanoma compared with untreated mice. However, despite the additive effect of TQ in addition to GK irradiation on B16-F10 melanoma cells in vitro, TQ did not add any significant survival benefit to GK treatment in mice with intracerebral melanoma. CONCLUSIONS: Our findings suggest that TQ would be a potential therapeutic agent in addition to GK to enhance the antitumor effect of irradiation. Further studies are required to support our findings.
Subject(s)
Apoptosis/drug effects , Benzoquinones/pharmacology , Brain Neoplasms/therapy , DNA Damage/drug effects , Melanoma, Experimental/therapy , Radiosurgery/methods , STAT3 Transcription Factor/drug effects , Actins/drug effects , Actins/metabolism , Actins/radiation effects , Animals , Apoptosis/radiation effects , Blotting, Western , Brain Neoplasms/secondary , Caspase 3/drug effects , Caspase 3/metabolism , Caspase 3/radiation effects , Cell Line, Tumor , Combined Modality Therapy , DNA Damage/radiation effects , In Vitro Techniques , Janus Kinase 2/drug effects , Janus Kinase 2/metabolism , Janus Kinase 2/radiation effects , Melanoma, Experimental/secondary , Mice , Phosphoproteins/drug effects , Phosphoproteins/metabolism , Phosphoproteins/radiation effects , Proto-Oncogene Proteins c-bcl-2/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/radiation effects , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/radiation effects , Survivin/drug effects , Survivin/metabolism , Survivin/radiation effects , bcl-2-Associated X Protein/drug effects , bcl-2-Associated X Protein/metabolism , bcl-2-Associated X Protein/radiation effectsABSTRACT
BACKGROUND: Prognosis of patients with melanoma brain metastasis is poor despite various chemotherapeutic agents. Researchers focus on finding effective treatment with a low risk of toxicity. Thymoquinone (TQ) has been found to be effective on different types of cancer. However, no data exist regarding the effect of TQ in intracerebral melanoma. The purpose of this study was to assess the effect of TQ in B16-F10 melanoma cell in vitro and intracerebral melanoma in vivo. METHODS: The mechanisms of efficacy were investigated using adenosine triphosphate assay for cytotoxicity, flow cytometry, and acridine orange staining for apoptosis, comet assay for genotoxicity, CM-H2DCF-DA (2,7-dichlorodihydrofluorescein) for intracellular reactive oxygen species (ROS) generation and ELISA methods for inflammatory cytokines. Western blotting was performed to assess the expressions of p-JAK2, p-STAT3, caspase-3, Bax, Bcl-2, and survivin. In addition, the effect of TQ was investigated in a model system of intracerebral melanoma in syngeneic mice. RESULTS: The median survival was improved by TQ in mice with intracerebral melanoma compared with the control group (16 days vs 9 days; P = 0.008). Cytotoxicity was enhanced by TQ in B16-F10 cells in a dose-dependent manner. TQ also induced apoptosis, DNA damage, and increased intracellular ROS. TQ inhibited p-STAT3, resulting in apoptosis through regulation of proapoptotic and antiapoptotic proteins. CONCLUSIONS: Our findings suggest that TQ would be an effective treatment in intracerebral metastatic lesions. This warrants further investigation.
