Sequential Dosing in Chemosensitization: Targeting the PI3K/Akt/mTOR Pathway in Neuroblastoma

Abstract

Breaking resistance to chemotherapy is a major goal of combination therapy in many tumors, including advanced neuroblastoma. We recently demonstrated that increased activity of the PI3K/Akt network is associated with poor prognosis, thus providing an ideal target for chemosensitization. Here we show that targeted therapy using the PI3K/mTOR inhibitor NVP-BEZ235 significantly enhances doxorubicin-induced apoptosis in neuroblastoma cells. Importantly, this increase in apoptosis was dependent on scheduling: while pretreatment with the inhibitor reduced doxorubicin-induced apoptosis, the sensitizing effect in co-treatment could further be increased by delayed addition of the inhibitor post chemotherapy. Desensitization for doxorubicin-induced apoptosis seemed to be mediated by a combination of cell cycle-arrest and autophagy induction, whereas sensitization was found to occur at the level of mitochondria within one hour of NVP-BEZ235 posttreatment, leading to a rapid loss of mitochondrial membrane potential with subsequent cytochrome c release and caspase-3 activation. Within the relevant time span we observed marked alterations in a ∼30 kDa protein associated with mitochondrial proteins and identified it as VDAC1/Porin protein, an integral part of the mitochondrial permeability transition pore complex. VDAC1 is negatively regulated by the PI3K/Akt pathway via GSK3β and inhibition of GSK3β, which is activated when Akt is blocked, ablated the sensitizing effect of NVP-BEZ235 posttreatment. Our findings show that cancer cells can be sensitized for chemotherapy induced cell death – at least in part – by NVP-BEZ235-mediated modulation of VDAC1. More generally, we show data that suggest that sequential dosing, in particular when multiple inhibitors of a single pathway are used in the optimal sequence, has important implications for the general design of combination therapies involving molecular targeted approaches towards the PI3K/Akt/mTOR signaling network.

Targeted Inhibition of Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Sensitizes Pancreatic Cancer Cells to Doxorubicin without Exacerbating Cardiac Toxicity

“…BEZ235 Augments Doxorubicin without Influencing Cardiotoxicity (Manara et al, 2010;Schult et al, 2012;Westhoff et al, 2013), we show for the first time that there are secondary effects of BEZ that can sensitize PDAC to DOX treatment. Our results demonstrate that BEZ cotreatment enhances DOX accumulation, leading to increased ROS formation and DNA damage.…”

“…It has previously been tested for use in hepatocellular carcinoma, multiple myeloma, and other cancers that commonly have mutations or increased gene expression that lead to overactivation of the PI3K pathway (Baumann et al, 2009;McMillin et al, 2009;Kirstein et al, 2013;Cebulla et al, 2015). BEZ has also been shown to have beneficial effects in combination with cytotoxic agents, including DOX (Manara et al, 2010;Schult et al, 2012;Westhoff et al, 2013). However, the effect of combination therapy in pancreatic cancer and on cardiotoxicity has not been reported.…”

Role of autophagy in the ω-3 long chain polyunsaturated fatty acid-induced death of lung cancer A549 cells

“…Coordinated inhibition of the mTOR and autophagy pathways promotes apoptosis and may be a novel therapeutic paradigm for the treatment of melanoma (36). Targeted therapy using the PI3K/mTOR inhibitor NVP-BEZ235 significantly enhances doxorubicin-induced apoptosis in neuroblastoma cells (37). However, certain studies report that activation of the PI3K/Akt/mTOR signaling pathway inhibits autophagy and induces cell death (38,39).…”

Oridonin enhances the anticancer activity of NVP-BEZ235 against neuroblastoma cells in vitro and in vivo through autophagy

“…SHSY-5Y cells at 5x10 6 suspended in 100 µl PBS were injected s.c. into the flank region of nude mice. When tumors were measurable and reached an average volume of 100 mm 3 , the mice were randomized into four groups for the following treatments: vehicle control, NVP-BEZ235 (20 mg/kg/day, i.p. ), oridonin (10 mg/ kg/day; i.p.…”

“…Neuroblastoma (NB) is a malignant paediatric tumor of the sympathetic nervous system that presents as a highly heterogeneous disease, ranging from spontaneous regression to high risk of fatality (1)(2)(3). Although the overall survival of neuroblastoma has increased greatly with the advances of diagnostic methods and therapeutic treatment over the recent years, the cure rate and life quality for high-risk neuroblastoma have only improved marginally when given aggressive conventional treatment (4,5).…”