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Supplementary Figure 2 from Regulation of Colorectal Cancer Cell Apoptosis by the n-3 Polyunsaturated Fatty Acids Docosahexaenoic and Eicosapentaenoic

Supplementary Figure 1 from Regulation of Colorectal Cancer Cell Apoptosis by the n-3 Polyunsaturated Fatty Acids Docosahexaenoic and Eicosapentaenoic

Mitochondrial Transcription Termination Factor 4 ( MTERF4 ) is a transcription factor involved in mitochondrial ribosomal biogenesis and was identified as a gene of interest via whole exome sequencing in a pediatric patient with hypertrophic cardiomyopathy (HCM) at Children’s Hospital of Wisconsin. The variants of interest identified in MTERF4 have not been previously reported in the literature or associated with HCM. Here, we combined precision genome editing via CRISPR/Cas9 system with human iPSC-derived cardiomyocytes (hiPSC-derived CMs) to model the HCM patient-specific MTERF4 variants. We hypothesize that MTERF4 variants are contributing to cardiomyocyte impairment, leading to the development of the hypertrophic phenotype. To improve our efficiency of CRISPR/Cas9 precision genome editing in hiPSCs, we are using a co-targeting with selection method. MTERF4 mutant clones and control hiPSCs (obtained from co-targeted but unmodified by CRISPR/Cas9) were identified by Sanger sequencing and assessed for pluripotency using immunostaining and gene expression. MTERF4 clones and controls were subjected to our modified Palecek matrix-overlay method for CM differentiation and evaluated at 3-4 weeks for cell size, mitochondrial function, and gene expression. The hiPSC-derived CMs cell size analysis by average pixel area of the MTERF4 mutant indicated that they are significantly (p=0.0012) larger compared to MTERF4 control. The assessment of mitochondrial function demonstrated that the maximal mitochondrial respiration may be reduced in MTERF4 mutant hiPSC-derived CMs compared to control (p=0.056). In known cardiovascular disease genes (NPPA, NPPB, GATA4, TNNT, MYL7, MYH7) associated with HCM, gene expression (qRT-PCR) were elevated. Preliminary data support our hypothesis through changes in CMs size, mitochondrial function, and transcriptional expression for one MTERF4 mutant clone and control. This approach has generated an in vitro tool to evaluate aspects diseases such as cardiomyopathy that can be used for diagnostic screening and therapy. Repeating our initial studies, and adding mechanistic studies, with additional MTERF4 mutant and control clones will further validate our hypothesis.

Several studies have suggested that the partially fermentable fibre Plantago ovata husk (PO) may have a protective effect on colorectal cancer (CRC). We studied the potentially pro-apoptotic effect of PO and the implicated mechanisms in CRC cells with different molecular phenotypes (Caco-2, HCT116, LoVo, HT-29, SW480) after PO anaerobic fermentation with colonic bacteria as it occurs in the human colon. The fermentation products of PO induced apoptosis in all primary tumour and metastatic cell lines, independent of p53, adenomatous polyposis coli, β-catenin or cyclo-oxygenase-2 status. Apoptosis was caspase-dependent and both intrinsic and extrinsic pathways were implicated. The intrinsic pathway was activated through a shift in the balance towards a pro-apoptotic environment with an up-regulation of B-cell lymphoma protein 2 homologous antagonist killer (BAK) and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) seen in HCT116 and LoVo cells. This resulted in mitochondrial membrane depolarisation, increased expression of caspase activators second mitochondria-derived activator of caspases (Smac)/Diablo, death effector apoptosis-inducing factor, apoptosome member apoptotic protease activating factor 1 and down-regulation of inhibitors of apoptosis Survivin and X-linked inhibitor of apoptosis in most cells. The extrinsic pathway was activated presumably through the up-regulation of death receptor (DR5). Some important differences were seen between primary tumour and metastatic CRC cells. Thus, metastatic PO-treated LoVo cells had a remarkable up-regulation of TNF-α ligand along with death-inducing signalling complex components receptor interacting protein and TNF-α receptor 1-associated death domain protein. The extrinsic pathway modulator FCICE-inhibitory protein (FLIP), an inhibitor of both spontaneous death ligand-independent and death receptor-mediated apoptosis, was significantly down-regulated after PO treatment in all primary tumour cells, but not in metastatic LoVo. These findings suggest that PO could potentially be a useful chemotherapy adjuvant.

Several studies have suggested that the n-3 fatty acids Docosahexaenoic (DHA) and Eicosapentaenoic (EPA) have an important protective effect on colorectal cancer, and this could be at least partly due to their proapoptotic activity. It is unclear, however, how this phenomenon is triggered and what mechanisms are implicated. Here, we show that both DHA and EPA have an important proapoptotic effect on colorectal cancer cells with different molecular phenotypes but not in noncancerous cells. Apoptosis is caspase dependent, and both intrinsic and extrinsic pathways are implicated. The dimerization of Bax and Bak, the depolarization of the mitochondrial membrane, and the subsequent release of cytochrome c and Smac/Diablo to the cytosol evidence the activation of the intrinsic pathway. The implication of the extrinsic pathway is shown by the activation of caspase-8, along with the down-regulation of FLIP. The timing of caspase-8 activation, and the oligomerization of Bid with Bax, suggest a cross-talk with the intrinsic pathway. None of the death receptors that commonly initiate the extrinsic pathway: FAS, TNF-R1, and TRAIL-R2 are found to be responsible for triggering the apoptosis cascade induced by DHA and EPA. Neither PPARγ nor cyclooxygenase-2, two likely candidates to regulate this process, play a significant role. Our findings suggest that the down-regulation of two key regulatory elements of the extrinsic and intrinsic pathways, FLIP and XIAP, respectively, is determinant in the induction of apoptosis by DHA and EPA. These fatty acids could potentially be useful adjuvant anticancer agents in combination with other chemotherapeutic elements.