Zhaoming Xu

Folate plays a role in the synthesis and repair of DNA and the generation of methyl groups. Folic acid (FA) is a synthetic oxidized form of folate used in food fortification and supplements in Canada. Increased colon cancer incidence has been correlated with FA fortification in several countries. The effect of FA on the development of colon cancer is controversial as other research shows a lack of association between FA fortification and colon cancer incidence. I hypothesize that FA affects proliferation and folate transporter expression in colon cancer cells differently than L-5-methyltetrahydrofolate (5MTHF). In addition, the forms of folate, reduced versus oxidized, would differentially affect the activity of the Wnt signaling pathway. The overall objective of my research is to investigate the effect of FA and 5MTHF on cell proliferation, the expression of selected folate transporters, and the activity of the Wnt signalling pathway in human colorectal adenocarcinoma (Caco2) cells. Caco2 cells were cultured for 3 or 5 days in folate-free RPMI 1640 medium supplemented with 10% dialyzed FBS and treated with 0, 0.9, 2.3, or 3.4 µM FA or MTHF. Cell viability was assessed using WST-1 colourimetric assay. Cell proliferation was assessed by BrdU colourimetric assay and cell cycle analysis with BrdU incorporation was measured by flow cytometry. The abundance of reduced folate transporter (RFC), folate receptor-α (FRα), proton-coupled folate transporter (PCFT), breast cancer resistance protein (BCRP) was assessed by Western blotting. β-Catenin nuclear localization was assessed by measuring the fluorescence of Alexa Fluor 488® using confocal microscopy. FA treatment increased cell proliferation compared to treatment with MTHF at all concentrations after 3 days. After 5 days, there was no difference in cell viability or cell proliferation. Cell cycle analysis after 5 days of 3.4 µM FA and 5MTHF treatment showed spikes in the pre-G1 phase compared to the control. Neither folate transporter expression nor β-Catenin nuclear localization was affected by FA and 5MTHF treatment under the conditions tested. This lack of effect of FA and 5MTHF on cell proliferation and the expression of selected folate transporters was possibly due to relatively short treatment duration.

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Zinc, an essential trace element for humans, exerts many physiological functions, including its indispensible role in growth. Dietary zinc deficiency in children results in growth retardation. However, the mechanism whereby zinc regulates growth remains unclear. MicroRNAs are a group of newly discovered, small, non-coding RNAs and have been demonstrated to play a regulatory role in cell proliferation. The expression of microRNA and process of producing mature microRNA can be influenced by cellular and tissue zinc status. Thus, the hypothesis for my research is that microRNA plays a role in mediating zinc-dependent cell proliferation. The overall objective was to determine whether microRNAs are involved in zinc-dependent cell proliferation in mouse fibroblast 3T3 cells.3T3 cells were cultured in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum (FBS) for 3 days. To deplete intracellular zinc, cells were cultured in the same media containing 5% (v/v) dimethyl sulfoxide (DMSO, control) or N,N,N’,N’- tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 2.5 μM) for 24 or 48 h with or without induction of quiescence (aphidicolin, 0.5 μg/ml; 24 h). To establish zinc-dependent effect, zinc was replenished at the final concentration of 0 (TPEN only), 1.25, 2.5 or 5 μM for 24 or 48 h. Cell proliferation was measured by cell cycle analysis using flow cytometry. MicroRNA expression profile was assessed by microRNA microarray.In the absence of quiescence induction, zinc-depletion for 24 or 48 h inhibited cell proliferation by 10.4% and 16.0% compared to control. In the presence of quiescence induction, zinc-depletion inhibited cell proliferation by 52.9%. Regardless of the status of quiescence, zinc replenishment at 1.25 μM nearly brought cell proliferation back to the level observed in the DMSO control, showing a zinc-dependent cell proliferation in 3T3 cells. Zinc-depletion increased the abundance of miR-132-3p, miR-212-3, and let-7e-3p, while zinc replenishment brought back the abundance of these three microRNAs to the level observed in the DMSO control. Interestingly, zinc-depletion decreased the abundance of miR-145b, and its abundance was increased after zinc replenishment. Overall, it appeared that microRNA played a role in zinc-mediated growth regulation in 3T3 cells; however, this role of microRNA remains to be affirmed by further investigation.

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Zinc is an essential trace element required for many physiological functions,including growth. At the cellular level, zinc is required for structural and catalytic roles inthousands of proteins, and adequate labile zinc is an important determinant of cellularviability. However, abnormal zinc accumulation in breast tissue is associated with breastcancer, suggesting that zinc status plays a role in breast cancer pathogenesis. Chelationinduceddepletion of labile intracellular zinc promotes apoptosis, or programmed cell death,in multiple breast cancer cell lines. The mechanisms whereby zinc regulates apoptosisremain unclear. In particular, little is known about the role of microRNAs (miRs), a novelclass of short non-coding RNA, involved in the regulation of gene expression. Zinc statuscan influence miR expression, and possibly the processing and stability of miRs. Thehypothesis of my thesis research is that miRs are involved in zinc depletion-inducedapoptosis in human breast cancer cells. The overall objective of this study was to determinethe involvement of miRs in zinc depletion-induced apoptosis in breast cancer MDA-MB-231cells. Zinc depletion for 24, 48 and 72 h induced apoptosis in 4.5, 24.4 and 28.0 % of thecells, respectively, indicating a time-dependent increase in zinc depletion-induced apoptosis.Expression of 8, 90, and 94 miRs were significantly altered during the early stages of zincdepletion-induced apoptosis, at 3, 12, and 24 h of zinc depletion, respectively. Overall,expression of 285 unique miRs was significantly affected by zinc depletion, duration of zincdepletion, and their interactions. qRT-PCR analysis confirmed that zinc depletion resulted inan increased abundance of miR-132-3p, miR-1246, miR-1273, miR-4484 and miR-4787-5pand a decreased abundance of miR-4521 in a time-dependent manner. MiR-132-3p and miR 1246 have previously been shown to play a role in mediating apoptosis in prostate cancerPC-3 and lung cancer A549 cells, respectively. In conclusion, abundance of numerous miRswas altered during the early stages of zinc depletion-induced apoptosis, indicating possibleinvolvement of these miRs in mediating zinc depletion-induced apoptosis. The role andtargets of these miRs in zinc depletion-induced apoptosis requires validation in furtherresearch.

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Retinoic acid (RA), a bioactive metabolite of vitamin A, inhibits growth in a variety of cancer cells including liver cancer. It is thought that this function of RA is achieved by modulating gene expression through complexing with retinoic acid receptors (RARs) and retinoid X receptors (RXRs), two groups of zinc-finger proteins. Zinc deficiency has been shown to affect gene expression and to impair DNA binding ability of zinc-finger proteins. The hypotheses of my thesis research were: 1) sufficient cellular zinc level is important for the effectiveness of RA-induced growth inhibition in hepatocarcinoma HepG2 cells; and 2) the influence of zinc on RA-induced growth inhibition is through modulating expression or function of RARs and RXRs, which in turn affects the expression of their target genes, CYP26a1 and RARβ. The overall objective was to examine the effects of zinc on RA-induced growth inhibition in HepG2 and the possible mechanisms involved. Zinc manipulation was achieved by culturing HepG2 cells for 6 d in low-zinc media supplemented with 0, 5, and 10 μmol/L zinc to mimic low-, adequate-, and high-zinc conditions. Growth in low-zinc media for 6 d reduced total cellular zinc and the labile intracellular pool of zinc by 29 and 86%, respectively. Treating the cells with 35 µM of RA for 12 h following zinc manipulation significantly reduced cell proliferation in all zinc-treatment groups compared to their corresponding RA control, with the greatest reduction in the high-zinc group. Cell cycle analysis showed that the proportion of cells in the S-phase was reduced by RA treatment at 24 and 72 h at all zinc levels, with the greatest reduction in cells cultured in high-zinc medium. Following growth in low-, adequate- and high-zinc medium, RA treatment elevated the abundance of RARβ and Cyp26a1 mRNA equally in all zinc-treatment groups compared to their correspondent RA controls. Growth in low zinc medium increased mRNA abundance of RXRα while RARα, RARβ, RARγ, RXRβ and RARγ were not affected. In conclusion, these results showed that increasing zinc appeared to sensitize HepG2 cells to RA-induced growth inhibition, but had no effect on RA-induced gene expression of CYP26a1 and RARβ.

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Breast cancer is the most frequently diagnosed cancer among Canadian women. Despite the use of advanced therapeutics, breast cancer remains the second leading cause of cancer death among Canadian women. Therefore, the development of novel and effective therapeutics to treat breast cancer remains an important goal. Defective or inhibited apoptosis is a major causative factor in the development and progression of cancer. Zinc is considered an apoptotic regulator. Further, previous work has also shown that there is an association between zinc depletion-induced apoptosis and an elevated intracellular Ca²⁺ level in human breast cancer MDA-MB-231 cells. Ca²⁺ is a known mediator of the mitochondrial apoptotic pathway. The overall objective of my thesis research was to investigate the role of intracellular Ca²⁺ and its involvement of the mitochondria in zinc depletion-induced apoptosis in human breast cancer MDA-MB-231 cells. MDA-MB-231 cells were cultured in DMEM containing FBS (10%) followed by depletion of intracellular zinc using N,N,N’,N’-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN; 20 µM) with or without the presence of intracellular Ca²⁺ chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid acetoxymethyl ester (BAPTA-AM; 10 or 20 µM). Apoptosis was assessed by caspase-9 and -3 activities using corresponding fluorogenic substrates and the proportion of cells with fragmented DNA using PI-staining flow cytometry assay. Intracellular Ca²⁺ was assessed using Fura-2 assay. Mitochondrial membrane potential was assessed by DiOC₆-staining flow cytometry assay. Cytochrome c release was detected by Western blot. Addition of TPEN resulted in an increase of caspase-9 and -3 activities, an increase in the proportion of cells with fragmented DNA, and a prolonged increase in intracellular Ca²⁺ level. TPEN treatment also reduced mitochondrial membrane potential and induced cytochrome c release. Zinc replenishment (10 – 40 µM) prevented TPEN-induced apoptosis. Intracellular Ca²⁺ chelation with BAPTA-AM suppressed TPEN-induced apoptosis, mitochondrial membrane potential loss, and cytochrome c release. Collectively these results showed that zinc depletion-induced apoptosis was mediated through the Ca²⁺-dependent mitochondrial apoptotic pathway in human breast cancer MDA-MB-231 cells.

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Breast cancer is the most prevalent type of cancer in Canadian women and ranks second in mortality. The cause of breast cancer deaths is the tumor growth in secondary locations. Zinc has been suggested to alter the affinity by which cells attach to the extracellular matrix. The strength of cell adhesion is paramount to the ability of cancerous cells to migrate. The hypothesis for my thesis research was that zinc promotes the metastasic potential of human breast cancer cells. The overall objective of my thesis research was to characterize the effects of zinc on the growth and metastatic potential of human breast cancer cells. Breast carcinoma MDA-MB-231 cells were cultured in DMEM plus 10% Chelex-100 treated FBS supplemented with 0 (zinc-deficient medium), 5 (zinc-adequate medium), or 25 (zinc-supplemented medium) µmol/L of Zn²⁺ as ZnSO₄. After culture for 96 h, the cells were harvested for determining total cellular zinc concentration, abundance of the labile intracellular pool of zinc, and cell growth. The metastatic potential of the cells was assessed by a combination of migration rate assessed using the wound-healing assay and migration distance using the single cell migration assay. The cells were also assessed for their adhesion on fibronectin, for the involvement of specific integrin subunits using blocking antibody, and integrin activation using FACS. Zinc treatments had no effect on total cellular zinc concentration, cell growth, and viability and essentially had no effect on abundance of the labile intracellular pool of zinc. Zinc as low as 5 µmol/L inhibited MDA-MB-231 cell migration on fibronectin, reduced magnesium-mediated promotion of adhesion and thus was likely involved in inhibiting magnesium-mediated integrin activation. With the use of blocking antibodies, it was determined that α5/β1 integrin was responsible for the adhesion of the cells to fibronectin and it was likely that zinc inhibited adhesion by blocking the activation of this specific form of integrin. Together these results suggested that zinc was an inhibitor of MDA-MB-231 migration on fibronectin, and thus likely an inhibitor of metastases, contrary to the hypothesis.

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