xav939 and Neuroblastoma

Neuroblastoma is one of the most common cancers in infancy, arising from the neuroblasts during embryonic development. This cancer is difficult to treat and resistance to chemotherapy is often found; therefore, clinical trials of novel therapeutic approaches, such as targeted-cancer signaling, could be an alternative for a better treatment. WNT signaling plays significant roles in the survival, proliferation, and differentiation of human neuroblastoma. In this report, WNT signaling of a malignant human neuroblastoma cell line, SH-SY5Y cells, was inhibited by XAV939, a specific inhibitor of the Tankyrase enzyme. XAV939 treatment led to the reduction of β-catenin within the cells, confirming its inhibitory effect of WNT. The inhibition of WNT signaling by XAV939 did not affect cell morphology, survival, and proliferation; however, the differentiation and sensitivity to anticancer drugs of human neuroblastoma cells were altered. The treatment of XAV939 resulted in the downregulation of mature neuronal markers, including β-tubulin III, PHOX2A, and PHOX2B, whereas neural progenitor markers (PAX6, TFAP2α, and SLUG) were upregulated. In addition, the combination of XAV939 significantly enhanced the sensitivity of SH-SY5Y and IMR-32 cells to doxorubicin in both 2D and 3D culture systems. Microarray gene expression profiling suggested numbers of candidate target genes of WNT inhibition by XAV939, in particular, p21, p53, ubiquitin C, ZBED8, MDM2, CASP3, and FZD1, and this explained the enhanced sensitivity of SH-SY5Y cells to doxorubicin. Altogether, these results proposed that the altered differentiation of human malignant neuroblastoma cells by inhibiting WNT signaling sensitized the cells to anticancer drugs. This approach could thus serve as an effective treatment option for aggressive brain malignancy.

Topics: Antineoplastic Combined Chemotherapy Protocols; beta Catenin; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Interactions; Drug Screening Assays, Antitumor; Gene Expression; Heterocyclic Compounds, 3-Ring; Humans; Molecular Targeted Therapy; Neuroblastoma; Spheroids, Cellular; Wnt Signaling Pathway

The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. The use of drugs inhibiting MGMT has been hindered by their haematologic toxicity and inefficiency. As a different strategy to inhibit MGMT we investigated cellular regulators of MGMT expression in multiple cancers. Here we show a significant correlation between Wnt signalling and MGMT expression in cancers with different origin and confirm the findings by bioinformatic analysis and immunofluorescence. We demonstrate Wnt-dependent MGMT gene expression and cellular co-localization between active β-catenin and MGMT. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs in mouse models. These findings have potential therapeutic implications for chemoresistant cancers, especially of brain tumours where the use of temozolomide is frequently used in treatment.

Topics: Animals; Antineoplastic Agents; Benzeneacetamides; beta Catenin; Brain Neoplasms; Camptothecin; Celecoxib; Cisplatin; Colorectal Neoplasms; Dacarbazine; DNA Modification Methylases; DNA Repair Enzymes; Doxorubicin; Drug Resistance, Neoplasm; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; Glucose-6-Phosphate Isomerase; Heterocyclic Compounds, 3-Ring; Humans; Immunoblotting; Immunohistochemistry; Irinotecan; Medulloblastoma; Mice; Neoplasm Transplantation; Neoplasms; Neuroblastoma; Pyrans; Pyrazines; Pyridines; Real-Time Polymerase Chain Reaction; Sulfones; Temozolomide; Triazoles; Tumor Suppressor Proteins; Vincristine; Wnt Proteins; Wnt Signaling Pathway

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. One fundamental issue regarding NB recurrence and metastasis is the maintenance of cancer stem cells (CSCs) stemness. Tankyrase 1 (TNKS1) is overexpressed in several types of cancers and in NB cell lines. XAV939 is a small molecule inhibitor of TNKS1 and can induce apoptosis of NB cells. In this study, we showed that the surface marker CD133 method was more suitable for isolating NB CSCs than the side-population method, and 60 µM etoposide was optimal for enriching NB CSCs. The NB CSCs were demonstrated in juvenescence or stemness state by electron microscopy, which was in line with the characteristics of CSCs. Furthermore, we demonstrated that the expression of the CSCs marker CD133 and migration ability of CSCs decreased after XAV939 treatment or by RNAi‑mediated knockdown of the TNKS1 gene. These findings suggest that XAV939 treatment or RNAi-TNKS1 inhibits the stemness and migration of NB CSCs via the repression of TNKS1, and TNKS1 may be a potential molecular target for eliminating NB CSCs by small molecule drugs.

Topics: AC133 Antigen; Antigens, CD; Cell Movement; Enzyme Inhibitors; Etoposide; Gene Expression Regulation, Neoplastic; Glycoproteins; Heterocyclic Compounds, 3-Ring; Humans; Neoplastic Stem Cells; Neuroblastoma; Peptides; Tankyrases; Tumor Cells, Cultured

Telomeres, telomerase and tankyrase (TNKS) have an extremely important and special association with human cell aging and cancer. Telomerase activity is abnormally high in cancer cells and is accompanied by the overexpression of tankyrase 1 (TNKS1). TNKS1 is a positive regulator of telomerase activation and telomere extension in the human body, indicating that TNKS1 may be a possible therapeutic target for cancer. XAV939 is a small-molecule inhibitor of TNKS1. The objective of the present study was to investigate the apoptotic effect of XAV939 on the neuroblastoma (NB) SH-SY5Y cell line, as well as the change in telomere length and telomerase activity and elucidate the mechanism from this perspective. In the present study, we initially treated SH-SY5Y cells with XAV939 and RNA interference (RNAi)-TNKS1, and subsequently chose the optimal sequence for RNAi-TNKS1. We then measured the telomere length using quantitative real-time polymerase chain reaction (qPCR) assay, detected the telomerase activity using the ELISA kit, observed apoptotic morphology by transmission electron microscopy, and detected the percentages of apoptotic cells using flow cytometry and Hoechst 33342 staining. We also determined the invasive ability by a cell invasion assay. The results showed that short hairpin RNA-2 (shRNA-2) was the optimal sequence for RNAi-TNKS1. Treatment with both XAV939 and RNAi-TNKS1 shortened the telomere length, promoted apoptosis and reduced the invasive ability of the SH-SY5Y cells, yet had no effect on telomerase activity. XAV939 promoted apoptosis and reduced the invasiveness of SH-SY5Y cells dependent on telomere shortening, and further research should be conducted to clarify the exact mechanisms. This research may contribute to the cure of malignant NB using multi-targeted therapy with small-molecule agents.

Topics: Apoptosis; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Heterocyclic Compounds, 3-Ring; Humans; Neuroblastoma; RNA, Small Interfering; Tankyrases; Telomere Shortening

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. The present treatment including surgery, chemotherapy and radiation, which have only 40% long-term cure rates, and usually cause tumor recurrence. Thus, looking for new effective and less toxic therapies has important significance. XAV939 is a small molecule inhibitor of tankyrase 1(TNKS1). The objective of this study is to investigate the effect of XAV939 on the proliferation and apoptosis of NB cell lines, and the related mechanism.. In the present study, we used both XAV939 treatment and RNAi method to demonstrate that TNKS1 inhibition may be a potential mechanism to cure NB. MTT method was used for determining the cell viability and the appropriate concerntration for follow-up assays. The colony formation assay, Annexin V staining and cell cycle analysis were used for detecting colony forming ability, cell apoptosis and the percentage of different cell cycle. The Western blot was used for detecting the expression of key proteins of Wnt/ beta-catenin (Wnt/β-catenin) signaling pathway.. The results showed that TNKS1 inhibition decreased the viability of SH-SY5Y, SK-N-SH and IMR-32 cells, induced apoptosis in SH-SY5Y as well as SK-N-SH cells, and led to the accumulation of NB cells in the S and G2/M phase of the cell cycle. Moreover, we demonstrated TNKS1 inhibition may in part blocked Wnt/β-catenin signaling and reduced the expression of anti-apoptosis protein. Finally, we also demonstrated that TNKS1 inhibition decreased colony formation in vitro.. These findings suggested that TNKS1 may be a potential molecule target for the treatment of NB.

Topics: Apoptosis; Cell Division; Cell Growth Processes; Cell Line, Tumor; Enzyme Inhibitors; G2 Phase; Heterocyclic Compounds, 3-Ring; Humans; Neuroblastoma; Tankyrases; Wnt Signaling Pathway

Neuroblastoma is the most common solid tumor in infancy. We have shown that the neuroblastoma cell line SK-N-SH contains CD133+ cells that are more resistant than 133- cells to Doxorubicin (DOX), a common chemotherapeutic agent. We hypothesize that activation of wnt signaling pathway in CD133+ cells contributes to their chemoresistance. To test this hypothesis, CD133+ cells were positively selected using magnetic micro-beads. Subsequently, CD133+ and negatively selected CD133- cells were treated with 100 ng/ml of DOX for up to 72 h. Then, cells were either lysed for total RNA extraction or fixed for immunostaining. Wnt "SIGNATURE" PCR Array was used to determine if changes in wnt related gene expression levels occurred and to estimate a pathway activity score. Expression of wnt pathway proteins β-Catenin and p-GSK3β (S-9) was determined by immunocytochemistry. Two wnt pathway inhibitors were used to determine the changes in cell viability, using the MTT assay. Results showed that wnt related genes were differentially expressed in CD133+ cells as compared to CD133- cells, both with and without DOX treatment. Pathway activity scores showed that DOX treatment significantly suppressed the wnt pathway activity in CD133- cells. Expression of β-catenin and p-GSK3β (S-9) was significantly greater in DOX treated and untreated CD133+ cells. The presence of wnt inhibitors with DOX decreased the number of live cells in CD133+ group and the percentage of live cells in both groups were equal. These data suggest that higher wnt pathway activity could be responsible for the chemoresistance of CD133+ cells in neuroblastoma cell lines.

Topics: AC133 Antigen; Antibiotics, Antineoplastic; Antigens, CD; beta Catenin; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Proliferation; Doxorubicin; Drug Resistance, Neoplasm; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Glycoproteins; Heterocyclic Compounds, 3-Ring; Humans; Immunoenzyme Techniques; Immunophenotyping; Neoplastic Stem Cells; Neuroblastoma; Peptides; Pyrimidinones; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Wnt Proteins; Wnt Signaling Pathway