Wing Pui Tsang

MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer

Background:MicroRNAs (miRNAs) are 19-25-nucleotides regulatory non-protein-coding RNA molecules that regulate the expressions of a wide variety of genes, including some involved in cancer development. In this study, we investigated the possible role of miR-143 in colorectal cancer (CRC).Methods:Expression levels of human mature miRNAs were examined using real-time PCR-based expression arrays on paired colorectal carcinomas and adjacent non-cancerous colonic tissues. The downregulation of miR-143 was further evaluated in colon cancer cell lines and in paired CRC and adjacent non-cancerous colonic tissues by qRT–PCR. Potential targets of miR-143 were defined. The functional effect of miR-143 and its targets was investigated in human colon cancer cell lines to confirm miRNA–target association.Results:Both real-time PCR-based expression arrays and qRT–PCR showed that miR-143 was frequently downregulated in 87.5% (35 of 40) of colorectal carcinoma tissues compared with their adjacent non-cancerous colonic tissues. Using in silico predictions, DNA methyltranferase 3A (DNMT3A) was defined as a potential target of miR-143. Restoration of the miR-143 expression in colon cell lines decreased tumour cell growth and soft-agar colony formation, and downregulated the DNMT3A expression in both mRNA and protein levels. DNMT3A was shown to be a direct target of miR-143 by luciferase reporter assay. Furthermore, the miR-143 expression was observed to be inversely correlated with DNMT3A mRNA and protein expression in CRC tissues.Conclusion:Our findings suggest that miR-143 regulates DNMT3A in CRC. These findings elucidated a tumour-suppressive role of miR-143 in the epigenetic aberration of CRC, providing a potential development of miRNA-based targeted approaches for CRC therapy.

Epigallocatechin gallate up-regulation of miR-16 and induction of apoptosis in human cancer cells

Epigallocatechin gallate (EGCG) is a major type of green tea polyphenols and is known to have cancer prevention effect. MicroRNAs (miRNAs) are 19 to 25 nucleotides and are believed to be important in gene regulation. In the present study, the influence of EGCG on the expressions of miRNAs in human cancer cells was investigated as this has not yet been reported. By miRNA microarray analysis, EGCG treatment was found to modify the expressions of some of the miRNAs in human hepatocellular carcinoma HepG2 cells, 13 were up-regulated and 48 were down-regulated. miR-16 is one of the miRNAs up-regulated by EGCG and one of its target genes is confirmed to be the anti-apoptotic protein Bcl-2. EGCG treatment induced apoptosis and down-regulated Bcl-2 in HepG2 cells. Transfection with anti-miR-16 inhibitor suppressed miR-16 expression and counteracted the EGCG effects on Bcl-2 down-regulation and also induction of apoptosis in cells. Results from the present study confirm the role of miR-16 in mediating the apoptotic effect of EGCG and also support the importance of miRNAs in the regulation of the biological activity of EGCG.

Let-7a microRNA suppresses therapeutics-induced cancer cell death by targeting caspase-3

MicroRNAs (miRNA) are endogenously expressed non-coding RNAs that regulate gene expression post-transcriptionally. Let-7a miRNA is a founding member in the let-7 family and its down-regulation in association with over-expression of RAS and HMGA2 oncogenes has previously been reported. In the present study, caspase-3, the executioner caspase, was confirmed to be the target of let-7a as ectopic expression of let-7a decreased the luciferase activity of a reporter construct containing the 3′ untranslated region of caspase-3 and at the same time repressed the enzyme expression in human squamous carcinoma A431 cells and hepatocellular carcinoma HepG2 cells. Moreover, let-7a was over-expressed while caspase-3 was down-regulated in A10A cells, a doxorubicin-resistant A431 subline. Enforced let-7a expression increased the resistance in A431 cells and HepG2 cells to apoptosis induced by therapeutic drugs such as interferon-gamma, doxorubicin and paclitaxel. On the other hand, down-regulation of let-7a by the anti-let-7a inhibitor increased the doxorubicin-induced apoptosis in A431 parent cells, A10A cells and HepG2 cells while the increase was suppressed by caspase-3 inhibitor. Both anti-let-7a inhibitor and caspase-3 inhibitor however failed to affect the drug-induced apoptosis in human breast cancer MCF7 cells, the cells that do not express caspase-3. Therefore, let-7a by targeting caspase-3 may play a functional role in modulating drug-induced cell death in human cancer cells.

p53-R273H gains new function in induction of drug resistance through down-regulation of procaspase-3

Development of drug resistance is one of the major obstacles in cancer chemotherapy. The molecular mechanism leading to drug resistance is still not fully understood. A10A cells, a doxorubicin-resistant subline of human squamous cell carcinoma A431 cells, showed cross-resistance to methotrexate and also resistance to the drug-induced apoptosis. The cells also showed overexpression of a mutated form of p53, p53-R273H (Arg to His at codon 273), and down-regulation of procaspase-3. Knockdown of p53-R273H by p53 small interfering RNA in A431 cells increased procaspase-3 level and sensitized the cells to drug-induced apoptosis. On the other hand, transfection of p53-R273H into p53 null human osteosarcoma Saos-2 cells down-regulated procaspase-3 level and induced resistance to the drug toxicity and drug-induced apoptosis. The results support the idea that p53-R273H may gain new functions in induction of drug resistance and impairment in drug-induced apoptosis through down-regulation of procaspase-3 level. The study sheds new light on the understanding of the gain of function and drug resistance mechanisms associated with mutant p53. [Mol Cancer Ther 2007;6(3):1054–8]

Downregulation of hepatoma-derived growth factor activates the Bad-mediated apoptotic pathway in human cancer cells

Hepatoma-derived growth factor (HDGF) is highly expressed in human cancer and its expression is correlated with poor prognosis of cancer. The growth factor is known to stimulate cell growth while the underlying mechanism is however not clear. Transfection with HDGF cDNA stimulated while its specific antisense oligonucleotides repressed the growth of human hepatocellular carcinoma HepG2 cells. Furthermore, knock-down of HDGF by antisense oligos also induced apoptosis in HepG2 cells and in other human cancer cells, e.g. human squamous carcinoma A431 cells. HDGF knock-down was found to induce the expression of the pro-apoptotic protein Bad and also inactivate ERK and Akt, which in turn led to dephosphorylation of Bad at Ser-112, Ser-136, and activation of the intrinsic apoptotic pathway, i.e. depolarization of the mitochondrial membrane, release of mitochondrial cytochrome c, increase in the processing of caspase 9 and 3. As HDGF knock-down not only suppresses the growth but also induces apoptosis in human cancer cells, HDGF may therefore serve as a survival factor for human cancer cells and a potential target for cancer therapy.

CD146+ Human Umbilical Cord Perivascular Cells Maintain Stemness under Hypoxia and as a Cell Source for Skeletal Regeneration

The human umbilical cord perivascular cells (HUCPVCs) have been considered as an alternative source of mesenchymal progenitors for cell based regenerative medicine. However, the biological properties of these cells remain to be well characterized. In the present study, HUCPVCs were isolated and sorted by CD146+ pericyte marker. The purified CD146+ HUCPVCs were induced to differentiate efficiently into osteoblast, chondrocyte and adipocyte lineages in vitro. Six weeks following subcutaneous transplantation of CD146+ HUCPVCs-Gelfoam-alginate 3D complexes in severe combined immunodeficiency (SCID) mice, newly formed bone matrix with embedded osteocytes of donor origin was observed. The functional engraftment of CD146+ HUCPVCs in the new bone regenerates was further confirmed in a critical-sized bone defect model in SCID mice. Hypoxic conditions suppressed osteogenic differentiation while increased cell proliferation and colony-forming efficiency of CD146+ HUCPVCs as compared to that under normoxic conditions. Re-oxygenation restored the multi-differentiation potential of the CD146+ HUCPVCs. Western blot analysis revealed an upregulation of HIF-1α, HIF-2α, and OCT-4 protein expression in CD146+ HUCPVCs under hypoxia, while there was no remarkable change in SOX2 and NANOG expression. The gene expression profiles of stem cell transcription factors between cells treated by normoxia and hypoxic conditions were compared by PCR array analysis. Intriguingly, PPAR-γ was dramatically downregulated (20-fold) in mRNA expression under hypoxia, and was revealed to possess a putative binding site in the Hif-2α gene promoter region. Chromatin immunoprecipitation assays confirmed the binding of PPAR-γ protein to the Hif-2α promoter and the binding was suppressed by hypoxia treatment. Luciferase reporter assay showed that the Hif-2α promoter activity was suppressed by PPAR expression. Thus, PPAR-γ may involve in the regulation of HIF-2α for stemness maintenance and promoting the expansion of CD146+ HUCPVCs in response to hypoxia. CD146+ HUCPVCs may serve as a potential autologous cell source for bone regeneration.

EPO Promotes Bone Repair through Enhanced Cartilaginous Callus Formation and Angiogenesis

Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPOs function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration.

Mechanistic Study on Growth Suppression and Apoptosis Induction by Targeting Hepatoma-derived Growth Factor in Human Hepatocellular Carcinoma HepG2 Cells

Hepatoma-derived growth factor (HDGF) is frequently overexpressed in human cancer. The growth factor was previously demonstrated to be a survival factor as knock-down of HDGF suppresses the growth and induces apoptosis in human cancer cells through the Bad-mediated intrinsic apoptotic pathway. However, inactivation of Bad cannot completely repress the apoptosis induced upon HDGF knock-down, indicating the presence of other unidentified pathways. In the present study, HDGF knock-down was shown to trigger the Fas-mediated extrinsic apoptotic pathway in human hepatocellular carcinoma HepG2 cells through NF-κB signaling pathway. Increases in Fas expression and fas promoter activity were detected upon HDGF knock-down by Western blot analysis and luciferase reporter assay. Knock-down of fas inhibited HDGF knock-down effect on apoptosis induction and growth suppression as revealed by annexin V binding assay and soft agar assay. Down-regulation of IκBα was also observed upon HDGF knock-down. Overexpression of IκBα by transient transfection or inhibition of NF-κB by BAY11-7082 suppressed HDGF knock-down effect on fas promoter activation, Fas up-regulation, apoptosis induction and growth suppression. Furthermore, the interaction of Fas-mediated extrinsic and Bad-mediated intrinsic apoptotic pathways was demonstrated as a stronger inhibition on apoptosis induction and growth suppression upon HDGF knock-down was observed when both pathways were inactivated. The results therefore suggested that, through both intrinsic and extrinsic apoptotic pathways, HDGF may function as a survival factor and be a potential target for cancer therapy.

Oncogene AF1q enhances doxorubicin-induced apoptosis through BAD-mediated mitochondrial apoptotic pathway.

AF1q is an oncogenic factor involved in leukemia development, thyroid tumorigenesis, and breast cancer metastasis. In the present study, AF1q was found to be down-regulated in a doxorubicin-resistant subline of human squamous carcinoma A431 cells. Knockdown of AF1q decreased the apoptosis induced by doxorubicin, Taxol, γ-radiation, IFN-α, and IFN-γ in A431 cells. On the other hand, overexpression of AF1q increased the doxorubicin-induced apoptosis in A431 cells as well as in HepG2 and HL60 cells. Both exogenous and ectopic expression of AF1q in A431 cells increased the mRNA and protein levels of BAD, a proapoptotic BCL-2 family protein. Gene silencing of BAD by small interfering RNA suppressed the AF1q enhancement of apoptosis, suggesting that BAD is downstream of AF1q in regulation of apoptosis. Furthermore, AF1q enhanced the mitochondrial membrane depolarization, mitochondrial cytochrome c release, and activation of caspase-9 and caspase-3 on doxorubicin treatment. Collectively, AF1q increases doxorubicin-induced apoptosis in cells through activation of BAD-mediated apoptotic pathway. The study provides the first evidence that AF1q plays a critical role in the regulation of apoptosis and drug resistance. [Mol Cancer Ther 2008;7(10):3160–8]

Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair

Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α) has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10−6 M) increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1) and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM) synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic marker genes including Mmp2, Mmp9, Mmp13, Adamts4 and Adamts5 was downregulated following Icariin treatment for 14 days. In a differentiation assay using bone marrow mesenchymal stem cells (MSCs) carrying HIF-1α floxed allele, the promotive effect of Icariin on chondrogenic differentiation is largely decreased following Cre recombinase-mediated deletion of HIF-1α in MSCs as indicated by Alcian blue staining for proteoglycan synthesis. In an alginate hydrogel 3D culture system, Icariin increases Safranin O positive (SO+) cartilage area. This phenotype is accompanied by upregulation of HIF-1α, increased proliferating cell nuclear antigen positive (PCNA+) cell numbers, SOX9+ chondrogenic cell numbers, and Col2 expression in the newly formed cartilage. Coincide with the micromass culture, Icariin treatment upregulates mRNA levels of Sox9, Col2α1, aggrecan and Col10α1 in the 3D cultures. We then generated alginate hydrogel 3D complexes incorporated with Icariin. The 3D complexes were transplanted in a mouse osteochondral defect model. ICRS II histological scoring at 6 and 12 weeks post-transplantation shows that 3D complexes incorporated with Icariin significantly enhance articular cartilage repair with higher scores particularly in selected parameters including SO+ cartilage area, subchondral bone and overall assessment than that of the controls. The results suggest that Icariin may inhibit PHD activity likely through competition for cellular iron ions and therefore it may serve as an HIF-1α activator to promote articular cartilage repair through regulating chondrocyte proliferation, differentiation and integration with subchondral bone formation.