Abubakar, Ibrahim Babangida
(2017)
In vitro investigation on synergistic anticancer effects between vitamin E isomers, pure compounds and crude alkaloid plant extracts on human cancerous cells.
PhD thesis, University of Nottingham.
Abstract
Anticancer chemotherapeutic treatment using single dose has been limited due to drug resistance and potential metabolic degradation. For instance, high-dose tocotrienols undergo metabolic degradation that limits the availability of therapeutic dose thereby limiting the potency in vivo. Combined treatment of tocotrienols at low dosage has been suggested as alternative to circumventing this limitation. This study was designed to investigate the cytotoxic effects and subsequently the apoptotic mechanisms of individual doses and combined treatments at lower dosages of tocotrienols (delta and gamma), jerantinines (A and B) and extracts (ethanol and alkaloid crude) from leaves and bark of Ficus hispida, Ficus fistulosa and Ficus schwarzii on lung (A549), brain (U87MG) and colon (HT-29) cancer cell lines. Neutral red uptake assay was conducted to evaluate the antiproliferative effect of individual and combined treatments. Staining techniques (histochemical and fluorescence), COMET assay flow cytometric analysis and immunofluorescence were conducted to evaluate cell morphology, DNA damage, cell cycle arrest pattern and antimicrotubule effects. Finally cell and molecular based assays were conducted to investigate the pathways for induction of apoptosis.
Cell viability study revealed that alkaloid crude extracts of leaves and bark of F. fistulosa demonstrated the highest potency with IC50 range of 0.96 – 46.81 µg/ml compared to F. schwarzii (8.79 – 107.9 µg/ml) and F. hispida (15.14 – 49.58 µg/ml) on A549, U87MG and HT-29 cells. Both delta- and gamma-tocotrienols induced antiproliferative effects on A549, U87MG and HT-29 cells with IC50 values of 3.12 - 12.40 µg/ml and 3.17 – 16.36 µg/ml, respectively. Potent antiproliferative effects were also evident for jerantinine A (IC50 0.62 – 1.74 µg/ml), jerantinine B (IC50 0.58 – 1.48 µg/ml) and vinblastine (IC50 0.03 – 0.71 µg/ml). However, similar toxic effects on these three compounds were also evident on non-cancerous lung fibroblast (MRC5) cells. The leaf and bark alkaloid crude extracts of F. fistulosa and F. schwarzii were selected for combined treatments. The combined treatment of IC20 doses of F. fistulosa with both delta- and gamma-tocotrienols induced synergistic antiproliferative effects (combination index (CI) < 1) on U87MG and HT-29 with up to 34.7-fold and 4-fold dose reductions for tocotrienols and F. fistulosa extracts, respectively. In contrast, additive (CI = 0.98) or antagonistic effects (CI > 1) were observed for IC20 doses of F. schwarzii extracts combined with delta- and gamma-tocotrienols on HT-29 cells. On the other hand, combined treatments of tocotrienols (delta and gamma) with IC20 doses of jerantinines (A and B) induced synergistic effects (CI < 1) on A549, U87MG and HT-29 cells causing up to 4.48-fold dose reduction of tocotrienols thus reducing toxicity towards MRC5 cells compared to cancer cells.
Further morphological and DNA damage assessment focusing on tocotrienols (delta and gamma), jerantinines (A and B) and combined low-dose treatments revealed anticancer features including cell shrinkage, nuclear chromatin condensation and fragmentation, membrane blebbing, apoptotic bodies and induction of predominantly double stranded DNA breaks. Cell cycle analysis demonstrated the induction of G0/G1 and G2/M cell cycle arrests by tocotrienols (delta and gamma) and jerantinines (A and B), respectively on U87MG, A549 and HT-29 cells. Meanwhile, G2/M (A549) and G0/G1 (U87MG and HT-29) cell cycle arrests were evident for combined low-dose treatments of tocotrienols (delta and gamma) with IC20 doses of jerantinines (A and B). Jerantinines A and B and combined low-dose treatments with tocotrienols (delta and gamma) caused disruption of microtubule networks and induction of caspase 8-, 9- and 3-mediated apoptosis with caspase-independent growth inhibition evidenced in the presence of caspase inhibitors on U87MG, A549 and HT-29 cells. In contrast, although treatments of tocotrienols (delta and gamma) alone caused similar apoptotic features as those of combined, disruption of microtubule networks were not characterized on these three cancer cell lines. Further mechanistic study on U87MG cells revealed that the apoptosis triggered by individual doses of tocotrienols (delta and gamma) involved the activation of TRAIL and Bid as well as the release of cytochrome C, thus confirming the recruitment of the death receptor and mitochondria-mediated pathways. On the other hand, individual doses of jerantinines (A and B) and combined low-dose treatments with tocotrienols resulted in the activation of TRAIL, FAS, p53 and Bid, as well as the release of cytochrome C. The activation of both death receptors, p53 and microtubule disruption by combined low-dose treatments demonstrates an improved mechanism of action comparing to individual doses of tocotrienols and jerantinines. In addition, the combined low-dose treatments also caused a reduction of required potent doses thereby minimizing the toxicity of jerantinines (A and B) towards the non-cancerous MRC5 cells. In conclusion, this research has presented valuable combined treatment candidates which are warranted for further investigations as future chemotherapeutic agents against cancers.
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