О проекте

Malignant glioma is an intractable cancer of the human brain. In spite of recent advances in medical technologies, malignant glioma is refractory to current and alternative therapies. The response of the tumor is very often quite different from what is observed in vitro, a probable reason being the culture conditions in vitro do not exactly match the in vivo conditions. In the first part of the thesis we have cultured cells in vitro under different conditions of cell density, oxygen and carbon dioxide concentration to mimic in vivo conditions and studied the influence of these conditions on the effect of curcumin. Curcumin has been described as a natural product and chemopreventive agent and has been tried in cancers. In this study we have looked at the effect of curcumin under different conditions of cell density [5,000 cells/well and 10,000 cells/well (96 well plates) for low and high cell density respectively], CO2 concentration (5% versus 10%) and O2 tension (18% versus 1.25%). Hence the initial experiments on curcumin response were done on different cell types U87MG, U373MG (glioblastoma cells having wild and mutated p53 respectively) as well as with SaOS-2 Osteosarcoma (null p53). We found that the most striking reversal of differences in cell response between normoxia and hypoxia in U373MG cells at low and high cell density in 5% CO2. In these conditions in low cell density the cells were more responsive to curcumin under normoxia but the reverse was true at high cell density (cells were more sensitive under hypoxia than normoxia). This effect has been studied in greater detail. The main action of curcumin is supposed to be via inhibition of NF-κB. The NF-κB reporter assay as well as the reduction of COX-2 and Bcl-2 broadly correlates with increased cell death in normoxia. The dose dependent increase in NF-κB reporter activity in hypoxic cells appears to be protective and hence provide a possible explanation as to why in vivo tumors with significant degree of hypoxia do not function as expected in monolayer culture experiments. Considering the extensive inter and intratumor variability of local microenvironment, it provides a basis for variations in response to curcumin during in vivo situations. In the second part of the thesis, the effect of hydroxylamine was studied under normoxia and hypoxia. Hydroxylamine has been used in vitro to modulate Reactive Oxygen Species (ROS) concentration and this has been used as an ROS enhancer. Our finding showed that there was significantly more cell killing (by MTT assay) in p53 mutated U373MG cells in normoxia as compared to hypoxia, which may because of less production of ROS (confirmed by flow cytometry using DCHF-DA). Apoptosis and cell cycle status by flow cytometry showed that under normoxic condition there was significantly increased apoptosis whereas under hypoxia it was less. xIAP mRNA gradually increased and COX-2 protein expression decreased under hypoxia as compared normoxia also revealed the same trend. The induction of apoptosis by hydroxylamine or other ROS enhancers under normoxia may be advantageous in cancer therapy. These finding suggests that this might, in future, enable us to have biochemical strategies to overcome resistance in tumors with significant components of hypoxia.

P “Modulation of curcumin responsive pathways by hypoxia: a study in glial cells”

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