Combinatorial application of tocotrienols and chemotherapeutic drugs enhances apoptosis and autophagy in colorectal cancer cells

Tham, Shiau Ying (2020) Combinatorial application of tocotrienols and chemotherapeutic drugs enhances apoptosis and autophagy in colorectal cancer cells. PhD thesis, University of Nottingham.

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Chemotherapy remains a cornerstone among the cancer treatment modalities but the therapeutic effectiveness of most conventional chemotherapeutic drugs is often limited by their dose-related toxicity. Tocotrienols, a class of vitamin E analogues, have been widely recognised as promising anti-cancer candidates due to their anti-proliferative, apoptotic, anti-invasive and anti-metastatic properties. Given that the multi-targeted actions of tocotrienols are suitable to be used as an adjunct treatment, a combinatory application with chemotherapeutic drugs in the cancer treatments may offer a synergism that provides augmented therapeutic effects and reduces high dose toxicities associated with chemotherapeutic drugs. Despite many combination studies with tocotrienols were conducted previously, the combined treatments of δ-tocotrienol (δT3) and conventional chemotherapeutic drugs, namely 5-fluorouracil (5FU) and doxorubicin (Dox) have not been reported in colorectal cancer cells. Hence, the current study aimed to study the therapeutic effects of δT3 + 5FU and δT3 + Dox combined treatments as an enhanced cancer therapeutic approach on Caco-2 and SW48 colorectal cells.

A high-throughput screening was initially conducted to identify synergistic combinations between tocotrienols and chemotherapeutic drugs. The selection of combination treatments was based upon the combination index (CI), drug reduction index (DRI), selectivity index (SI) and/or the capacity for inhibiting of clonogenic survival. Current study identified that synergisms are present on Caco-2 (CI= 0.45, DRI= 16.25, SI=21.88) and SW48 (CI=0.72, DRI=3.54, SI=5.58) colorectal cancer cells receiving δT3 + 5FU combined treatment. Meanwhile, δT3 + Dox combined treatment resulted a synergism on Caco-2 (CI=0.75, DRI, 2.80, SI=21.44) and a slight antagonism on SW48 (CI=1.50, DRI=0.95, SI=56.55). Nonetheless, both combinations exerted significant anti-clonogenic survivals on Caco-2 and SW48 cells, which warrants further investigations.

The ideal combined treatments were identified and subjected to various assays in order to investigate for their apoptosis-inducing properties, which include phosphatidylserine externalisation, DNA damage, cell cycle perturbation and DNA fragmentation. The presence of apoptosis was confirmed morphologically by the presence of cell shrinkage, membrane blebbing, nuclear condensation and fragmentation. Enhanced apoptosis and DNA fragmentation were detected in both cell lines receiving δT3 + 5FU and δT3 + Dox combined treatments as compared to their individual single treatments. The cellular apoptotic effect induced by δT3 + 5FU combined treatment was mediated through enhanced single-stranded DNA breaks and S-phase arrest. In contrast, δT3 + Dox combined treatment caused enhanced single-stranded and double-stranded DNA breaks and G2/M phase arrest.

To study the underlying apoptosis mechanism, several apoptosis-related proteins family such as caspases, inhibitor of apoptosis (IAP) and B-cell lymphoma 2 (Bcl-2) were evaluated. The combined treatments resulted in significantly higher caspase-3 activation than the individual single treatments, accompanied by the downregulation of IAP proteins (e.g., cIAP1, survivin and XIAP), suggesting the occurrence of a chemosensitisation effect toward apoptosis. Despite clear caspase-8 and -3 activations in response to δT3 + 5FU combined treatment was found, only SW48 but not Caco-2 cells exhibited caspase-3 dependent apoptosis, suggesting an additional cell death pathway was involved. The upregulation of Bax and/or downregulation in Bcl-2 have suggested an involvement of mitochondrial outer membrane permeabilisation. The findings of cyclosporine A inhibited the mitochondrial permeability transition and successfully prevented cell death, signify the importance of mitochondria in shaping cell death induced by the combined treatments. The results collectively suggest an involvement of caspase-dependent apoptosis and caspase-independent cell death.

In order to detect the autophagy manifestation induced by the combined treatments, acridine orange and monodansylcadaverine (MDC) staining methods were performed. The autophagy-related proteins namely microtubule-associated protein light chain 3 (LC3)-II and beclin-1 were also studied. Pharmacological inhibition of autophagy was performed using 3-methyladenine (3-MA) and bafilomycin A1 (Baf-1) autophagy inhibitors to delineate the role of autophagy in relation to cell death. An enhanced autophagy was also detected in the combined treatments as evidenced by significantly higher MDC intensity and elevated LC3-II protein. Inhibition of autophagy by 3-MA and Baf-1 successfully prevented cell death for Caco-2 and SW48 cell lines, could be an indication that an autophagic cell death has been involved.

In conclusion, both δT3 + 5FU and δT3 + Dox combined treatments in the study have been shown executing multi-targeted actions in killing the colorectal cancer cells. Therefore, it could potentially offer valuable therapeutic effects for the colorectal cancer treatment in a near future. Yet, further mechanistic, in vivo and clinical investigations of these combined treatments are still necessary to materialize its cancer treatment application.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Loh, Sandy Hwei San
Mai, Chun Wai
Fu, Ju Yen
Keywords: tocotrienol, 5-fluorouracil, doxorubicin, colorectal cancer, apoptosis, autophagy
Subjects: R Medicine > RC Internal medicine
Faculties/Schools: University of Nottingham, Malaysia > Faculty of Science and Engineering — Science > School of Biosciences
Item ID: 59416
Depositing User: THAM, SHIAU YING
Date Deposited: 22 Feb 2020 04:40
Last Modified: 21 Feb 2022 04:30

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