PURPOSE: We adopted a combinatorial approach with the joint application of γ-tocotrienol and jerantinine A at lower concentrations in order to minimize toxicity towards non-cancerous cells while improving the potency on brain cancer cells.
METHODS: The antiproliferative potency of individual γ-tocotrienol and jerantinine A as well as combined in low-concentration was firstly evaluated on U87MG cancer and MRC5 normal cells. Morphological changes, DNA damage patterns, cell cycle arrests and the effects of individual and combined low-concentration compounds on microtubules were then investigated. Finally, the potential roles of caspase enzymes and apoptosis-related proteins in mediating the apoptotic mechanisms were investigated using apoptosis antibody array, ELISA and Western blotting analysis.
RESULTS: Combinatorial study between γ-tocotrienol at a concentration range (0-24µg/ml) and fixed IC20 concentration of jerantinine A (0.16µg/ml) induced a potent antiproliferative effect on U87MG cells and led to a reduction on the new half maximal inhibitory concentration of γ-tocotrienol (i.e.tIC50=1.29µg/ml) as compared to that of individual γ-tocotrienol (i.e. IC50=3.17µg/ml). A reduction on undesirable toxicity to MRC5 normal cells was also observed. G0/G1 cell cycle arrest was evident on U87MG cells receiving IC50 of individual γ-tocotrienol and combined low-concentration compounds (1.29µg/ml γ-tocotrienol + 0.16µg/ml jerantinine A), whereas, a profound G2/M arrest was evident on cells treated with IC50 of individual jerantinine A. Additionally, individual jerantinine A and combined compounds (except individual γ-tocotrienol) caused a disruption of microtubule networks triggering Fas- and p53-induced apoptosis mediated via the death receptor and mitochondrial pathways.
CONCLUSIONS: These findings demonstrated that the combined use of lower concentrations of γ-tocotrienol and jerantinine A induced potent cytotoxic effects on U87MG cancer cells resulting in a reduction on the required individual concentrations and thereby minimizing toxicity of jerantinine A towards non-cancerous MRC5 cells as well as probably overcoming the high-dose limiting application of γ-tocotrienol. The multi-targeted mechanisms of action of the combination approach have shown a therapeutic potential against brain cancer in vitro and therefore, further in vivo investigations using a suitable animal model should be the way forward.
MATERIAL AND METHODS: A) Effects of various doses of nicotine on in vitro embryonic development: Female mice were treated with 1.0, 3.0, or 5.0 mg/kg/day nicotine for 7 consecutive days. Animals were superovulated, cohabited overnight, and sacrificed. Embryos were cultured in vitro. Plasma was assayed. B) Effects of concomitant treatment of nicotine concurrently with various doses of gamma-TCT on in vitro embryonic development: Female mice were treated with nicotine (5.0 mg/kg/day), gavaged gamma-TCT of 30, 60, or 90 mg/kg/day or nicotine concurrently with gamma-TCT of 3 different doses for 7 consecutive days. Animals were superovulated, cohabited overnight, and sacrificed. Embryos were cultured and plasma was assayed.
RESULTS: A) Effects of various doses of nicotine on in vitro embryonic development: Number of hatched blastocysts decreased in 1.0 and 3.0 mg/kg/day nicotine groups. Nicotine at 5.0 mg/kg/day stopped embryo development at morula. MDA concentrations increased following all nicotine doses. B) Effects of concomitant treatment of nicotine concurrently with various doses of gamma-TCT on in vitro embryonic development: Embryo development was completed in all groups. MDA concentration increased only in the group treated with nicotine concurrently with 30 mg/kg/day gamma-TCT.
CONCLUSIONS: Nicotine impairs in vitro embryo development and increases MDA in plasma. The deleterious impact of nicotine on embryo development is reversed by supplementing gamma-TCT concurrently with nicotine.