Enhanced inhibition of clonogenic survival of human medulloblastoma cells by multimodal treatment with ionizing irradiation, epigenetic modifiers, and differentiation-inducing drugs

Abstract

BackgroundMedulloblastoma (MB) is the most common pediatric brain tumor. Current treatment regimes consisting of primary surgery followed by radio- and chemotherapy, achieve 5-year overall survival rates of only about 60 %. Therapy-induced endocrine and neurocognitive deficits are common late adverse effects. Thus, improved antitumor strategies are urgently needed. In this study, we combined irradiation (IR) together with epigenetic modifiers and differentiation inducers in a multimodal approach to enhance the efficiency of tumor therapy in MB and also assessed possible late adverse effects on neurogenesis.MethodsIn three human MB cell lines (DAOY, MEB-Med8a, D283-Med) short-time survival (trypan blue exclusion assay), apoptosis, autophagy, cell cycle distribution, formation of gH2AX foci, and long-term reproductive survival (clonogenic assay) were analyzed after treatment with 5-aza-2′-deoxycytidine (5-azadC), valproic acid (VPA), suberanilohydroxamic acid (SAHA), abacavir (ABC), all-trans retinoic acid (ATRA) and resveratrol (RES) alone or combined with 5-aza-dC and/or IR. Effects of combinatorial treatments on neurogenesis were evaluated in cultured murine hippocampal slices from transgenic nestin-CFPnuc C57BL/J6 mice. Life imaging of nestin-positive neural stem cells was conducted at distinct time points for up to 28 days after treatment start.ResultsAll tested drugs showed a radiosynergistic action on overall clonogenic survival at least in two-outof-three MB cell lines. This effect was pronounced in multimodal treatments combining IR, 5-aza-dC and a second drug. Hereby, ABC and RES induced the strongest reduction of clongenic survival in all three MB cell lines and led to the induction of apoptosis (RES, ABC) and/or autophagy (ABC). Additionally, 5-aza-dC, RES, and ABC increased the S phase cell fraction and induced the formation of gH2AX foci at least in oneout-of-three cell lines. Thereby, the multimodal treatment with 5-aza-dC, IR, and RES or ABC did not change the number of normal neural progenitor cells in murine slice cultures.ConclusionIn conclusion, the radiosensitizing capacities of epigenetic and differentiation-inducing drugs presented here suggest that their adjuvant administration might improve MB therapy. Thereby, the combination of 5-aza-dC/IR with ABC and RES seemed to be the most promising to enhance tumor control without affecting the normal neural precursor cells.

Efficient cell death induction in human glioblastoma cells by photodynamic treatment with Tetrahydroporphyrin-Tetratosylat (THPTS) and ionizing irradiation

“…To detect DNA double strand breaks (DSB), immunfluorescence staining of phosphorylated (Ser139) histone H2AX (gammaH2AX), an early DNA repair protein, was performed as described previously [ 24 ]. Therefore, GBM cells were seeded in 8-well chamber slides (2000–2500 cells/ well), allowed to adhere for 24 h, and irradiated (4 Gy) or THPTS-PDT treated, except for untreated controls.…”

“…Clonogenic assay was performed as previously described [ 24 ]. Submaximal THPTS concentrations were used to enable detection of combined effects with irradiation or of repetitive effects.…”

Application of Small Epigenetic Modulators in Pediatric Medulloblastoma

“…Interestingly, also the combination of VPA or vorinostat with irradiation showed similar effects compared to decitabine/irradiation treatments on the mentioned cell lines, even though to a lower extent (42). The latter most powerful combination deserves in vivo validation.…”

Intracellular AFP Blocks All-trans retinoic acid induced ATG7 expression and Autophagy in Hepatocellular carcinoma cells

“…In in ATRA induced autophagy [14,[21][22][23][24]. In other cell types, including several other solid tumor types, ATRA was also able to induce autophagy [15,25]. In these studies, expression alterations of certain ATGs or signaling molecules were always displayed as the underlying mechanisms, which seemingly was not powerful enough to establish direct links between ATRA and autophagy, as the involvement and the function of RAR always lacked.…”