Ya-Lan Wang

PARP and PARG Inhibitors—New Therapeutic Targets in Cancer Treatment

Today, the number of cancer patients throughout the world is increasing alarmingly and as per the World Health Organisation (WHO) data and statistics the prediction for the year 2020 will be 15 million new cases as compared to only 10 million cases in year 2000 leaving us dumbfounded. A lot of effort has been put in by researchers and scientists over decades to find drugs helpful in the treatment of cancers for the benefit of patients—The latest being the Poly ADP-ribose polymerase (PARP) and the Poly ADP-ribose glycohydrolase (PARG) inhibitors. This review highlights their mechanism of action under the rationale of their use and current development in the field of cancer.

Poly(ADP-Ribose) Polymerase Inhibition Down-Regulates Expression of Metastasis-Related Genes in CT26 Colon Carcinoma Cells

Objectives: The current study was designed to test the hypothesis that inhibition of poly(ADP-ribose) polymerase in colorectal cancer mediates down-regulation of metastasis-related gene expression through the regulation of nuclear factor-κB (NF-κB) activity. Methods: Mouse colon carcinoma cells (CT26) were treated with and without the PARP inhibitor 5-aminoisoquinolin-1(2H)-one hydrochloride (5-AIQ). We investigated adhesion, migration and invasion of differently treated CT26 cells. In addition, the expression levels of PARP, NF-κB, integrin β1, MMP-9 and MMP-2 as well as the activities of NF-κB, MMP-9 and MMP-2 were determined by Western blot, electrophoretic mobility gel shift assay and zymography, respectively. Results: Inhibition ofPARP attenuated the adhesion of CT26 cells to the extracellular matrix and their migration and invasion through Matrigel. In addition, the results of Western blot showed that the expression levels of PARP, NF-κB, integrin β1, MMP-9 and MMP-2 were reduced in 5-AIQ-treated CT26 cells; the activities of NF-κB, MMP-9 and MMP-2 were also suppressed. Conclusions: Inhibition of PARP down-regulates the expression of metastasis-related genes in mouse colon carcinoma cells. This could be, at least in part, through the regulation of NF-κB activity, but the precise mechanisms of action remain to be elucidated.

Silencing Poly (ADP-Ribose) Glycohydrolase (PARG) Expression Inhibits Growth of Human Colon Cancer Cells In Vitro via PI3K/Akt/NFκ-B Pathway

Poly ADP-ribose polymerase (PARP) which is closely related to Poly ADP-ribose glycohydrolase (PARG) has already been thoroughly investigated in both experimental and clinical cancer trials compared to the latter. Nevertheless, in this experiment the importance of PARG expression was highlighted; whereby it is being silenced via lentivirus vector-mediated short hairpin RNA (shRNA). MTT assay showed that there was an inhibition in human Lovo colon cancer cell growth and flow cytometry demonstrated an increase in the population of cells in G0/G1 phase with a decrease in the S phase in transfected Lovo cells. Furthermore, our results suggested that the effect of silencing PARG leads to the inhibition of PARP expression; related to a decrease in the expression of Nuclear Factor Kappa-B (NFκ-B) with an increase in Akt473 phosphorylation; suggesting that the Phosphoinositol 3-kinase (PI3K)/Akt/NFκ-B pathway is important for cellular growth and proliferation. Hence, this study emphasizes and converges on the relevance of silencing PARG which inhibits growth of human colonic cancer cells via PI3K/Akt/NFκ-B pathway; as colon carcinoma remains to be amongst one of the commonest cancers throughout the world with high morbidity and mortality rates.

Inhibition of arginine ADP-ribosyltransferase 1 reduces the expression of poly(ADP-ribose) polymerase-1 in colon carcinoma

Poly(ADP-ribose) polymerase-1 (PARP-1) which mediates poly-ADP-ribosylation, has been extensively investigated in carcinoma compared to arginine ADP-ribosyltransferase 1 (ART1), which mediates mono‑ADP‑ribosylation. Previous studies have demonstrated that these enzymes promote proliferation and tumor development in colon carcinoma. However, whether there is any association between PARP-1 and ATR1 in colon carcinoma, remains unelucidated. In this study, using immunohistochemical analysis, we detected a higher expression of PARP-1 and ART1 in 63 samples from patients with colon carcinoma compared to 10 samples of normal colonic mucosa; our results revealed a positive correlation between the expression of PARP-1 and ART1 in the 63 human colon carcinoma tissue samples. To determine the correlation between PARP-1 and ART1, inhibitors of PARP-1 and ART1 and lentivirus vector‑mediated ART1 short‑hairpin RNA (shRNA) were used to culture CT26 murine colon adenocarcinoma cells separately. Using double‑label immunofluorescence assay, we detected the expression of PARP-1 in the CT26 cells, which was decreased following treatment with 5‑aminoisoquinolinone (5-AIQ, a PARP-1 inhibitor) or meta‑iodobenzylguanidine (MIBG, an ART1 inhibitor). However, the expression of ART1 only decreased when the CT26 cells were treated with MIBG. Furthermore, our results demonstrated that silencing ART1 inhibited PARP‑1 expression by decreasing the expression of nuclear factor-κB (NF-κB), inhibiting ras homolog A (RhoA). Hence, our data demonstrate the positive correlation between ART1 and PARP-1; the inhibition of ART1 activity downregulates PARP-1 expression by decreasing the activity of NF-κB in CT26 colon carcinoma cells.

ART1 silencing enhances apoptosis of mouse CT26 cells via the PI3K/Akt/NF-κB pathway.

Background/Aims: Colorectal carcinoma is one of the most common cancers world-wide, with high morbidity and mortality rates. Arginine ADP-ribosyltransferase 1(ART1) is an important ecto-ADP-ribose transferase and has been proven to be intimately involved in a number of biological processes. However, the influence of ART1 on survival and apoptosis of colorectal carcinoma cells and the potential mechanism of action of ART1 remain uncharacterized. Methods: ART1 was silenced via lentiviral vector-mediated short hairpin RNA (shRNA) in CT26 colon carcinoma cells, and cisplatin (CDDP) was applied to induce apoptosis. Survival and apoptosis rate of CT26 cells was assessed by CCK8 assay, flow cytometry and Hoechst 33342 staining. Expression and activity of signaling proteins were detected by Western blot. Results: ART1 knockdown enhanced the inhibition of cell survival and increased the apoptosis induced by CDDP. Furthermore, the reduced survival rate correlated with reduced levels of phos-AktThr308 and phos-IκBa and reduced NF-κB p65 nuclear translocation. A decline in Bcl-2 and Bcl-xl expression and an increase in Bax expression may explain the enhanced apoptosis. Conclusion: This study provides a molecular mechanism for the function of ART1 in colorectal carcinoma and defines a potential therapeutic target for the enhanced treatment of this prominent world-wide disease.

Novel hydrophilic docetaxel (CQMU-0519) analogue inhibits proliferation and induces apoptosis in human A549 lung, SKVO3 ovarian and MCF7 breast carcinoma cell lines.

Objectives of this investigation were not merely to perform a comparative study with original docetaxel, but to define anti‐proliferative and apoptotic effects of novel hydrophilic docetaxel (CQMU‐0519) analogue on A549 lung, SKVO3 ovary and MCF7 breast carcinoma cell lines.

Synergistic effect of arginine-specific ADP-ribosyltransferase 1 and poly(ADP-ribose) polymerase-1 on apoptosis induced by cisplatin in CT26 cells

Arginine-specific ADP-ribosyltransferase 1 (ART1) and poly(ADP-ribose) polymerase-1 (PARP-1) are both post‑translational modification proteins. Inhibition of PARP1 induces apoptosis in cancer cells, and ART1 regulates RhoA which promotes apoptosis in hepatic cancer cells when inhibited. However, the interaction of ART1 and PARP-1 on the effect of apoptosis has not yet been elucidated. In the present study, lentiviral vector-mediated ART1-cDNA was transfected into CT26 cells, and the apoptosis rate was detected by flow cytometric assay and Hoechst 33342 staining. Relevant factors were detected by reverse transcriptase-PCR and western blotting. The results showed that the apoptosis rate in the ART1-cDNA CT26 cells treated with PARP-1 inhibitor 5-aminoisoquinoline (5-AIQ) and cisplatin increased, when compared with the ART1-cDNA CT26 cells treated with cisplatin only or the untreated ART1-cDNA CT26 cells. Further studies have shown that PARP-1 is in the downstream of ART1, and plays a role in ART1-mediated CT26 cell apoptosis through the ROCK1/NF-κB/PARP-1 pathway when induced by cisplatin. We also found that in cisplatin-treated cells, activated caspase 3 cleaved PARP-1 and the decreased level of PARP-1 in turn decreased the expression of nuclear factor (NF)-κB, Cox-2 and increased caspase 3, resulting in the enhanced ability of ART1 to regulate CT26 cell apoptosis. Our research provides initial sight into the synergistic effect of ART1 and PARP-1 on apoptosis induced by cisplatin in murine colon carcinoma CT26 cells.

Arginine ADP-ribosyltransferase 1 promotes angiogenesis in colorectal cancer via the PI3K/Akt pathway

Arginine adenosine diphosphate (ADP)-ribosyltransferase 1 (ART1) is known to play an important role in many physiological and pathological processes. Previous studies have demonstrated that ART1 promotes proliferation, invasion and metastasis in colon carcinoma. However, it was unclear whether ART1 is involved in angiogenesis in cases of colorectal cancer (CRC). In the present study, lentiviral vector-mediated ART1-cDNA or ART1-shRNA were transfected into LoVo cells, and the LoVo cells transfected with ART1-cDNA or ART1-shRNA were co-cultured with human umbilical vein endothelial cells (HUVECs) to determine the influence of ART1 on HUVECs. The proliferation, migration and angiogenesis of HUVECs were monitored using a cell counting kit-8 assay, a Transwell migration assay and immunohistochemical analysis in intrasplenic allograft tumors, respectively. Hypoxia-inducible factor 1-α (HIF-1α), total (t-) Akt, phosphorylated (p-)Akt, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression levels were detected via western blot analysis. Our results revealed that HUVECs which were co-cultured with ART1-cDNA LoVo cells showed higher proliferation, migration and angiogenic abilities, but a reduction was noted in those cultured with ART1-shRNA LoVo cells; p-Akt, HIF-1α, VEGF and bFGF expression was increased in HUVECs cultured with ART1-cDNA-transfected LoVo cells, but reduced in ART1-shRNA-transfected LoVo cells. In a mouse xenograft model, we noted that the tumor microvessel density (MVD) was significantly increased in intrasplenic transplanted ART1-cDNA CT26 tumors but decreased in intrasplenic transplanted ART1-shRNA tumors. These data suggest that ART1 promoted the expression of HIF-1α via the Akt pathway in tumor cells. It also upregulated VEGF and bFGF and enhanced angiogenesis in HUVECs. Thus, we suggest that ART1 plays an important role in the invasion of CRC cells and the metastasis of CRC.

Novel Hydrophilic Taxane Analogues Inhibit Growth of Cancer Cells

In our era there has been several anti-cancer drugs which have undergone both experimental and clinical trials; however, due to their poor solubilities, numerous side effects, insufficient bioavailability and poor compliance, many have resulted into poor outcomes. Therefore, our aim was to investigate the effects of novel hydrophilic taxanes analogues CQMU-0517 and CQMU-0519 on growth of A549 lung, SKVO3 ovary and MCF7 breast carcinoma cell lines. Different concentrations of original paclitaxel, CQMU-0517, original docetaxel and CQMU-0519 were utilized on three cell lines, where cell growth was assessed using cell culture kit-8 and flow cytometry analysis. The results unveiled that CQMU-0517 and CQMU-0519 suppressed cell growth in the three particular cell lines, cell cycle arrest being evident in the G2/M phase. Hence, the results showed that these new taxane analogues have potential and warrant future clinical trials.

Filtrating colorectal cancer associated genes by integrated analyses of global DNA methylation and hydroxymethylation in cancer and normal tissue.

Recently, 5-hydroxymethylcytosine patterning across the tumor genome was considered as a hallmark of cancer development and progression. However, locus-specific difference of hydroxymethylation between colorectal cancer and normal tissue is unknown. In this study, we performed a newly developed method, HMST-seq, to profile 726 aberrant methylated loci and 689 aberrant hydroxymethylated loci synchronously in genome wide of colorectal cancers, majority of which presented higher methylation or lower hydroxymethylationin than in normal group. Besides, abnormal hydroxymethylated modification was more frequently occur at proximal regions close to TSSs and TSSs regions than abnormal methylation. Subsequently, we screened four genes (ALOX15, GHRHR, TFPI2 and TKTL1) with aberrant methylation and aberrant hydroxymethylation at some genome position by functional enrichment analysis as candidate genes associated with colorectal cancer. Our results may allow us to select differentially epigenetically modified target genes implicated in colorectal cancer tumorigenesis.