Using a microplate spectrophotometer (Wallac 1420 Victor 2; PerkinElmer, Waltham, MA), fluorescence was detected for alamarBlue assays (excitation, 560 nm; emission, 590 nm) or transferred to a white-walled 96-well microplate to measure luminescence for Caspase-Glo 3/7 assays. we show that allosteric AKT inhibition in these cells induces cytoprotective autophagy. Inhibition of autophagy using chloroquine (CQ) alone or in combination with Akti-1/2 leads to a significant decrease Gefarnate in viable cell number. In fact, Akti-1/2 sensitizes EOC cells to CQ-induced cell death by exhibiting markedly reduced EC50 values in combination-treated cells compared with CQ alone. In addition, we evaluated the effects of the novel (Spautin-1) and demonstrate that Spautin-1 inhibits autophagy in a Beclin-1-impartial manner in primary EOC cells and cell lines. Multicellular EOC spheroids are highly sensitive to Akti-1/2 and CQ/Spautin-1 cotreatments, but resistant to each agent alone. Indeed, combination index analysis revealed strong synergy between Akti-1/2 and Spautin-1 when both brokers were used to affect cell viability; Akti-1/2 and CQ cotreatment also displayed synergy in most samples. Taken together, we propose that combination AKT inhibition and autophagy blockade would show efficacious to reduce residual EOC cells for supplying ovarian cancer recurrence. Introduction The need for new and more effective therapeutics in ovarian cancer is usually highlighted by the low rate of survival experienced by patients with this disease. For women with localized disease who are treated surgically, 5-12 months survival is usually 90% (1). The vast majority of patients, however, present with metastatic disease characterized by widespread intraperitoneal dissemination (1). Although debulking surgery and chemotherapy can be initially effective Gefarnate at reducing tumor burden in these patients (2), advanced-stage epithelial ovarian cancer (EOC) has a high rate of disease recurrence and, as a consequence, a dramatically reduced 5-12 months survival rate of only 27.3% (1). Over the last 20 years, treatment strategies for metastatic EOC have remained largely unchanged, therefore new and complementary therapeutics are needed to provide greater survival benefit to ovarian cancer patients. To this end, numerous targeted therapies are being developed and are currently undergoing clinical trials in ovarian cancer. Brokers such as bevacizumab and olaparib that exploit alterations in angiogenesis and DNA damage responses pathways, respectively, have both demonstrated promising improvements in progression-free survival (3C5). Inhibitors of PI3K/AKT/mammalian target of rapamycin (mTOR) signaling are also being pursued since this pathway exhibits activating alterations in a large proportion of high-grade serous ovarian tumors (6,7). Clinical trials of such brokers, however, have proved disappointing thus far in ovarian cancer. For example, inhibition of epidermal growth factor receptor family members epidermal growth factor receptor and ErbB2/HER2 have yielded overall response rates of only 0C7% (8C10). Likewise, a phase II trial of the mTORC1 inhibitor temsirolimus showed insufficient benefit in progression-free survival to warrant subsequent phase III study (11). The failure of such brokers in ovarian cancer is probably a complex phenomenon attributable to many factors; we hypothesize that one key factor is the cellular survival mechanism known as autophagy. Macroautophagy (herein referred to as autophagy) is usually a conserved self-digestion mechanism that functions at basal levels in eukaryotic cells to maintain homeostasis and promote survival under conditions of cellular stress (12C14). Autophagy can also be induced by numerous anticancer brokers, especially those that target the PI3K/AKT/mTOR pathway and result in the inhibition of mTORC1, the canonical autophagy repressor (15,16). Therapy-induced autophagy has been shown to promote tumor cell survival (17C19), thereby blunting the effectiveness of anticancer brokers. Given that phase I/II trials of novel PI3K/AKT/mTOR pathway inhibitors are currently underway in ovarian cancer (clinicaltrials.gov), it is essential to determine whether tumor cells subjected to this class of inhibitors upregulate autophagy as a cytoprotective response. If this is the case, a novel therapeutic strategy may involve combining PI3K/AKT/mTOR pathway inhibitors with autophagy inhibitors to minimize the tumor cytoprotective response and maximize therapeutic efficacy. Our studies focused on metastatic high-grade serous ovarian cancer, utilizing cell cultures derived from patient ascites (fluid in the peritoneal cavity that accumulates as a result IL-2 antibody of metastatic disease) (20). Ascites contains single tumor cells and multicellular aggregates or spheroids (21C23), the dissemination of which throughout the peritoneal cavity is usually thought to seed secondary metastases (21,22,24,25). Thus, we utilized both non-adherent spheroid cultures and traditional Gefarnate adherent monolayers in our studies. We demonstrate that although AKT inhibition.
