Targeting DNA-PKcs and telomerase in brain tumour cells

Abstract

BackgroundPatients suffering from brain tumours such as glioblastoma and medulloblastoma have poor prognosis with a median survival of less than a year. Identifying alternative molecular targets would enable us to develop different therapeutic strategies for better management of these tumours.MethodsGlioblastoma (MO59K and KNS60) and medulloblastoma cells (ONS76) were used in this study. Telomerase inhibitory effects of MST-312, a chemically modified-derivative of epigallocatechin gallate, in the cells were assessed using telomere repeat amplification protocol. Gene expression analysis following MST-312 treatment was done by microarray. Telomere length was measured by telomere restriction fragments analysis. Effects of MST-312 on DNA integrity were evaluated by single cell gel electrophoresis, immunofluorescence assay and cytogenetic analysis. Phosphorylation status of DNA-PKcs was measured with immunoblotting and effects on cell proliferation were monitored with cell titre glow and trypan blue exclusion following dual inhibition.ResultsMST-312 showed strong binding affinity to DNA and displayed reversible telomerase inhibitory effects in brain tumour cells. In addition to the disruption of telomere length maintenance, MST-312 treatment decreased brain tumour cell viability, induced cell cycle arrest and double strand breaks (DSBs). DNA-PKcs activation was observed in telomerase-inhibited cells presumably as a response to DNA damage. Impaired DNA-PKcs in MO59J cells or in MO59K cells treated with DNA-PKcs inhibitor, NU7026, caused a delay in the repair of DSBs. In contrast, MST-312 did not induce DSBs in telomerase negative osteosarcoma cells (U2OS). Combined inhibition of DNA-PKcs and telomerase resulted in an increase in telomere signal-free chromosomal ends in brain tumour cells as well. Interestingly, continual exposure of brain tumour cells to telomerase inhibitor led to population of cells, which displayed resistance to telomerase inhibition-mediated cell arrest. DNA-PKcs ablation in these cells, however, confers higher cell sensitivity to telomerase inhibition, inducing cell death.ConclusionsEfficient telomerase inhibition was achieved with acute exposure to MST-312 and this resulted in subtle but significant increase in DSBs. Activation of DNA-PKcs might indicate the requirement of NHEJ pathway in the repair telomerase inhibitor induced DNA damage. Therefore, our results suggest a potential strategy in combating brain tumour cells with dual inhibition of telomerase and NHEJ pathway.

Spheroid-Derived Cells From Renal Adenocarcinoma Have Low Telomerase Activity and High Stem-Like and Invasive Characteristics

“…It has been suggested that CSCs could be sensitive to targeted therapies against telomerase (54). MST-312 is a derivative of a component of green tea and a novel telomerase inhibitor with potential therapeutic activity (55,56). MST-312 does not inhibit the growth of normal cells but effectively inhibits metastatic cancer cells (56).…”

MST-312 induces G2/M cell cycle arrest and apoptosis in APL cells through inhibition of telomerase activity and suppression of NF-κB pathway

“…As compared with EGCG, MST-312 possesses prominent advantages in terms of chemical stability, the effective dose for the induction of telomere shortening, and the circumvention of acquired resistance [32]. Although previous studies have shown that MST-312 inhibits telomerase activity in tumor cells [32][33][34], its mode of mechanism is yet to be fully understood.…”

MST-312 Alters Telomere Dynamics, Gene Expression Profiles and Growth in Human Breast Cancer Cells

“…Furthermore, it is suggested that the rapid proliferation rate of cancer cells could make them more susceptible to crisis following telomerase inhibition compared to germline cells with normal telomerase activity [13]. Several studies have shown that MST-312 inhibits telomerase activity and induces growth arrest selectively in tumor cells [14,15,16]. MST-312, an analogue of the green tea epigallocatechin gallate (EGCG), provides a chemically more stable alternative EGCG while retaining the ability to inhibit telomerase activity.…”