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BLM C Bleomycin. Similar to lipoxin A4, resolvins and protectins, even prostaglandins (PGE1, PGE2, PGF2 and PGI2) tested both in the pre- and simultaneous treatment schedules did not influence the growth inhibitory action of bleomycin on IMR-32 cells but never studied under the same conditions the effect of other fatty acids such as GLA, DGLA, AA, ALA, EPA and DHA and prostaglandins, leukotrienes and thromboxanes. DiHDPA) and MT-DADMe-ImmA 10(S),17(S)-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid (protectin: 10(S),17(S)DiHDoHE), metabolites of DHA, significantly inhibited the growth of IMR-32 cells. Pre-treatment with AA, GLA, DGLA and EPA and simultaneous treatment with all PUFAs used in the study augmented growth inhibitory action of bleomycin. Surprisingly, both indomethacin and nordihydroguaiaretic acid (NDGA) at 60 and 20 g/ml respectively enhanced the growth of IMR-32 cells even in the presence of bleomycin. AA enhanced oxidant stress in IMR-32 cells as evidenced by an increase in lipid peroxides, superoxide dismutase levels and glutathione peroxidase activity. These results suggest that PUFAs suppress growth of human neuroblastoma cells, augment growth inhibitory action of bleomycin by enhancing formation of lipid peroxides and altering the status of anti-oxidants and, in all probability, increase the formation of lipoxins, resolvins and protectins from their respective precursors that possess growth inhibitory actions. Introduction Previously, we and others showed that several polyunsaturated fatty acids (PUFAs) have selective cytotoxic action on many tumor cells of different types with little or no action on normal cells [1]C[14]. But, PUFAs themselves are not very effective in eliminating cancer cells in an situation partly, due to the fact that they are tightly bound to albumin and other proteins and hence, are unavailable to bring about their tumoricidal action [15]C[17]. Furthermore, PUFAs may be metabolized into several eicosanoids that may have other unwanted actions. Hence, it is desirable to develop methods whereby PUFAs are selectively delivered to tumor cells to produce their anti-cancer actions and/or given in combination with anti-cancer drugs so that the combined anti-cancer drug(s)+PUFAs may have a significant cytotoxic MT-DADMe-ImmA action on cancer cells compared to either agent alone. Studies showed that indeed a combination of PUFAs and conventional anti-cancer drugs have more potent action on tumor cells compared to either compound alone [18]C[23]. Some studies suggested that this tumoricidal action of PUFAs is not dependent Rabbit polyclonal to ISYNA1 on the formation of cyclo-oxygenase (COX) and lipoxygenase (LOX) products though, this has been disputed [1], [2], [24]C[28]. This uncertainty of the involvement of COX and LOX products on the growth/apoptosis of tumor cells is usually further supported by the observation that different prostaglandins either enhance or inhibit growth depending on the dose and type of the compounds tested and much less is known about the action of leukotrienes and thromboxanes on cancer cells [29]C[42]. In this context, it is noteworthy that effect MT-DADMe-ImmA of lipoxins derived from AA; resolvins from EPA and DHA and protectins from DHA around the growth of tumor cells has not been well evaluated though some studies did suggest that they may have anti-proliferative properties [43]C[47]. Many of these studies did not evaluate direct action of prostaglandins, leukotrienes, lipoxins, resolvins and protectins around the growth of tumor cells and much less is known about the effect of pre- and simultaneous treatment of tumor cells with PUFAs and their eicosanoid products around the anti-proliferative action of conventional anti-cancer drugs. In the present study, we evaluated the effect of various PUFAs, prostaglandins, leukotrienes, lipoxins, resolvins and protectins around the proliferation of human neuroblastoma (IMR-32) cells and compared these results to those obtained with COX and LOX inhibitors. The modulatory influence of PUFAs, prostaglandins, leukotrienes, lipoxins, resolvins and protectins on bleomycin-induced growth inhibitory action on IMR-32 cells was also studied. Finally, we evaluated the effect of AA, as a representative of unsaturated lipids, and bleomycin on anti-oxidant content, formation of lipid peroxides and nitric oxide in IMR-32 cells. Materials and Methods Reagents All culture media and additives were purchased from Sigma Aldrich Chemicals Pvt. Ltd., Bangalore, India. Bleomycin was purchased from Cipla, Goa, India. All PUFAs and their metabolites (Prostaglandins, Leukotrienes, Lipoxin A4, Protectins and Resolvins) used in the present study were purchased from Cayman Chemical Company, Michigan, USA. Cell culture conditions Human neuroblastoma cell line (IMR-32) obtained from Center for Cellular and Molecular Biology, Hyderabad, India (origin of source, ATCC) was grown in DMEM (pH 7.4) supplemented with bicarbonate, 100 U/ml penicillin, 100 g/ml streptomycin, 1.25 g/ml amphotericin B, 10% FBS at 37C with 5% CO2. IMR-32 grows as a monolayer and was subcultured when they became confluent. For culture experiments, cells were harvested from the confluent flask by washing them with phosphate buffered saline (PBS, pH 7.4) and treating with Trypsin (0.25%) C EDTA (0.02%) for 3 minutes. Trypsin was immediately inactivated by addition of equal volume of FBS and centrifuged to pellet the cells which were used.AA enhanced oxidant stress in IMR-32 cells as evidenced by an increase in lipid peroxides, superoxide dismutase levels and glutathione peroxidase activity. bleomycin. Surprisingly, both indomethacin and nordihydroguaiaretic acid (NDGA) at 60 and 20 g/ml respectively enhanced the growth of IMR-32 cells even in the presence of bleomycin. AA enhanced oxidant stress in IMR-32 cells as evidenced by an increase in lipid peroxides, superoxide dismutase levels and glutathione peroxidase activity. These results suggest that PUFAs suppress growth of human neuroblastoma cells, augment growth inhibitory action of bleomycin by enhancing formation of lipid peroxides and altering the status of anti-oxidants and, in all probability, increase the formation of lipoxins, resolvins and protectins from their respective precursors that possess growth inhibitory actions. Introduction Previously, we and others showed that several polyunsaturated fatty acids (PUFAs) have selective cytotoxic action on many tumor cells of different types with little or no action on normal cells [1]C[14]. But, PUFAs themselves are not very effective in eliminating cancer cells in an situation partly, due to the fact that they are tightly bound to albumin and other proteins and hence, are unavailable to bring about their tumoricidal action [15]C[17]. Furthermore, PUFAs may be metabolized into several eicosanoids that may have other unwanted actions. Hence, it is desirable to develop methods whereby PUFAs are selectively delivered to tumor cells to produce their anti-cancer actions and/or given in combination with anti-cancer drugs so that the combined anti-cancer drug(s)+PUFAs may have a significant cytotoxic action on cancer cells compared to either agent alone. Studies showed that indeed a combination of PUFAs and conventional anti-cancer drugs have more potent action on tumor cells compared to either compound alone [18]C[23]. Some studies suggested that the tumoricidal action of PUFAs is not dependent on the formation of cyclo-oxygenase (COX) and lipoxygenase (LOX) products though, this has been disputed [1], [2], [24]C[28]. This uncertainty of the involvement of COX and LOX products on the growth/apoptosis of tumor cells is further supported by the observation that different prostaglandins either enhance or inhibit growth depending on the dose and type of the compounds tested and much less is known about the action of leukotrienes and thromboxanes on cancer cells [29]C[42]. In this context, it is noteworthy that effect of lipoxins derived from AA; resolvins from EPA and DHA and protectins from DHA on the growth of tumor cells has not been well evaluated though some studies did suggest that they may have anti-proliferative properties [43]C[47]. Many of these studies did not evaluate direct action of prostaglandins, leukotrienes, lipoxins, resolvins and protectins on the growth of tumor cells and much less is known about the effect of pre- and simultaneous treatment of tumor cells with PUFAs and their eicosanoid products on the anti-proliferative action of conventional anti-cancer drugs. In the present study, we evaluated the effect of various PUFAs, prostaglandins, leukotrienes, lipoxins, resolvins and protectins on the proliferation of human neuroblastoma (IMR-32) cells and compared these results to those obtained with COX and LOX inhibitors. The modulatory influence of PUFAs, prostaglandins, leukotrienes, lipoxins, resolvins and protectins on bleomycin-induced growth inhibitory action on IMR-32 cells was also studied. Finally, we evaluated the effect of AA, as a representative of unsaturated lipids, and bleomycin on anti-oxidant content, formation of lipid peroxides and nitric oxide in IMR-32 cells. Materials and Methods Reagents All culture media and additives were purchased from Sigma Aldrich Chemicals Pvt. Ltd., Bangalore, India. Bleomycin was purchased from Cipla, Goa, India. All PUFAs MT-DADMe-ImmA and their metabolites (Prostaglandins, Leukotrienes, Lipoxin MT-DADMe-ImmA A4, Protectins and Resolvins) used in the present study were purchased from Cayman Chemical Company, Michigan, USA. Cell culture conditions Human neuroblastoma cell line (IMR-32) obtained from Center for Cellular and Molecular Biology, Hyderabad, India (origin of source, ATCC) was grown in DMEM (pH 7.4) supplemented with bicarbonate, 100 U/ml penicillin, 100 g/ml streptomycin, 1.25 g/ml amphotericin B, 10% FBS at 37C with 5% CO2. IMR-32 grows as a monolayer and was subcultured when they became confluent. For culture experiments, cells were harvested from the confluent flask by washing them with phosphate buffered saline (PBS, pH 7.4) and treating with Trypsin (0.25%) C EDTA (0.02%) for 3 minutes. Trypsin was immediately inactivated by addition of equal volume of FBS and centrifuged to pellet the cells which were used for various studies as described below. Effect of bleomycin on the proliferation of IMR-32 cells (Fig. 9). Similarly, both resolvins and protectins also did not produce.