Enhancing radiation response by a second-generation TRAIL receptor agonist using a new in vitro organoid model system.

BACKGROUND: For many cancer types, including colorectal carcinoma (CRC), combined modality treatments have shown to improve outcome, but are frequently associated with significant toxicity, illustrating the need for new therapeutic approaches. Based on preclinical data, TRAIL receptor agonists appeared to be promising agents for cancer therapy especially in combination with DNA damaging regimens. Here, we present the combination of the second-generation TRAIL receptor agonist APG-880 with radiation in a new and clinically relevant 3D model system. METHODS: To investigate the effect of APG-880 in combination with radiation we performed short-term cytotoxicity and long-term clonogenic survival assays in established CRC cell lines, and in tumor organoids derived from colon cancer patients. RESULTS: APG-880 is a potent inducer of apoptosis in CRC cell lines and in patient-derived CRC organoids. Furthermore, a supra-additive effect on cytotoxicity was found when APG-880 and radiation were combined simultaneously, with combination indices around 0.7. Lastly, in the long-term survival assays, we demonstrated a radiosensitizing effect of APG-880 with dose enhancement factors between 1.3 and 1.5. CONCLUSIONS: In a new, clinically relevant CRC-organoid model system we demonstrated a more than additive combined effect between the second-generation TRAIL receptor agonist APG-880 and radiation.

Targeting anti-apoptotic Bcl-2 by AT-101 to increase radiation efficacy: data from in vitro and clinical pharmacokinetic studies in head and neck cancer.

BACKGROUND: Pro-survival Bcl-2 family members can promote cancer development and contribute to treatment resistance. Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by overexpression of anti-apoptotic Bcl-2 family members. Increased levels of these anti-apoptotic proteins have been associated with radio- and chemoresistance and poor clinical outcome. Inhibition of anti-apoptotic Bcl-2 family members therefore represents an appealing strategy to overcome resistance to anti-cancer therapies. The aim of this study was to evaluate combined effects of radiation and the pan-Bcl-2 inhibitor AT-101 in HNSCC in vitro. In addition, we determined human plasma levels of AT-101 obtained from a phase I/II trial, and compared these with the effective in vitro concentrations to substantiate therapeutic opportunities. METHODS: We examined the effect of AT-101, radiation and the combination on apoptosis induction and clonogenic survival in two HNSCC cell lines that express the target proteins. Apoptosis was assessed by bis-benzimide staining to detect morphological nuclear changes and/or by propidium iodide staining and flow-cytometry analysis to quantify sub-diploid apoptotic nuclei. The type of interaction between AT-101 and radiation was evaluated by calculating the Combination Index (CI) and by performing isobolographic analysis. For the pharmacokinetic analysis, plasma AT-101 levels were measured by HPLC in blood samples collected from patients enrolled in our clinical phase I/II study. These patients with locally advanced HNSCC were treated with standard cisplatin-based chemoradiotherapy and received dose-escalating oral AT-101 in a 2-weeks daily schedule every 3 weeks. RESULTS: In vitro results showed that AT-101 enhances radiation-induced apoptosis with CI's below 1.0, indicating synergy. This effect was sequence-dependent. Clonogenic survival assays demonstrated a radiosensitizing effect with a DEF37 of 1.3 at sub-apoptotic concentrations of AT-101. Pharmacokinetic analysis of patient blood samples taken between 30 min and 24 h after intake of AT-101 showed a dose-dependent increase in plasma concentration with peak levels up to 300-700 ng/ml between 1.5 and 2.5 h after intake. CONCLUSION: AT-101 is a competent enhancer of radiation-induced apoptosis in HNSCC in vitro. In addition, in vitro radiosensitization was observed at clinically attainable plasma levels. These finding support further evaluation of the combination of AT-101 with radiation in Bcl-2-overexpressing tumors.

NAD⁺ depletion by APO866 in combination with radiation in a prostate cancer model, results from an in vitro and in vivo study.

BACKGROUND: APO866 is a highly specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), inhibition of which reduces cellular NAD(+) levels. In this study we addressed the potential of NAD(+) depletion as an anti-cancer strategy and assessed the combination with radiation. METHODS: The anticipated radiosensitizing property of APO866 was investigated in prostate cancer cell lines PC3 and LNCaP in vitro and in PC3 xenografts in vivo. RESULTS: We show that APO866 treatment leads to NAD(+) depletion. Combination experiments with radiation lead to a substantial decrease in clonogenic cell survival in PC3 and LNCaP cells. In PC3 xenografts, treatment with APO866 resulted in reduced intratumoral NAD(+) levels and induced significant tumor growth delay. Combined treatment of APO866 and fractionated radiation was more effective than the single modalities. Compared with untreated tumors, APO866 and radiation alone resulted in tumor growth delays of 14 days and 33 days, respectively, whereas the combination showed a significantly increased tumor growth delay of 65 days. CONCLUSIONS: Our studies show that APO866-induced NAD(+) depletion enhances radiation responses in tumor cell survival in prostate cancer. However, the in vitro data do not reveal a solid cellular mechanism to exploit further clinical development at this moment.

Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells.

S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK (extracellular-signal-regulated kinase)/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, the results of the present study suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus.

AT-101, a small molecule inhibitor of anti-apoptotic Bcl-2 family members, activates the SAPK/JNK pathway and enhances radiation-induced apoptosis.

BACKGROUND: Gossypol, a naturally occurring polyphenolic compound has been identified as a small molecule inhibitor of anti-apoptotic Bcl-2 family proteins. It induces apoptosis in a wide range of tumor cell lines and enhances chemotherapy- and radiation-induced cytotoxicity both in vitro and in vivo. Bcl-2 and related proteins are important inhibitors of apoptosis and frequently overexpressed in human tumors. Increased levels of these proteins confer radio- and chemoresistance and may be associated with poor prognosis. Consequently, inhibition of the anti-apoptotic functions of Bcl-2 family members represents a promising strategy to overcome resistance to anticancer therapies. METHODS: We tested the effect of (-)-gossypol, also denominated as AT-101, radiation and the combination of both on apoptosis induction in human leukemic cells, Jurkat T and U937. Because activation of the SAPK/JNK pathway is important for apoptosis induction by many different stress stimuli, and Bcl-X(L) is known to inhibit activation of SAPK/JNK, we also investigated the role of this signaling cascade in AT-101-induced apoptosis using a pharmacologic and genetic approach. RESULTS: AT-101 induced apoptosis in a time- and dose-dependent fashion, with ED50 values of 1.9 and 2.4 microM in Jurkat T and U937 cells, respectively. Isobolographic analysis revealed a synergistic interaction between AT-101 and radiation, which also appeared to be sequence-dependent. Like radiation, AT-101 activated SAPK/JNK which was blocked by the kinase inhibitor SP600125. In cells overexpressing a dominant-negative mutant of c-Jun, AT-101-induced apoptosis was significantly reduced. CONCLUSION: Our data show that AT-101 strongly enhances radiation-induced apoptosis in human leukemic cells and indicate a requirement for the SAPK/JNK pathway in AT-101-induced apoptosis. This type of apoptosis modulation may overcome treatment resistance and lead to the development of new effective combination therapies.

Alkylphospholipids inhibit capillary-like endothelial tube formation in vitro: antiangiogenic properties of a new class of antitumor agents.

Synthetic alkylphospholipids (APLs), such as edelfosine, miltefosine and perifosine, constitute a new class of antineoplastic compounds with various clinical applications. Here we have evaluated the antiangiogenic properties of APLs. The sensitivity of three types of vascular endothelial cells (ECs) (bovine aortic ECs, human umbilical vein ECs and human microvascular ECs) to APL-induced apoptosis was dependent on the proliferative status of these cells and correlated with the cellular drug incorporation. Although confluent, nondividing ECs failed to undergo apoptosis, proliferating ECs showed a 3-4-fold higher uptake and significant levels of apoptosis after APL treatment. These findings raised the question of whether APLs interfere with new blood vessel formation. To test the antiangiogenic properties in vitro, we studied the effect of APLs using two different experimental models. The first one tested the ability of human microvascular ECs to invade a three-dimensional human fibrin matrix and form capillary-like tubular networks. In the second model, bovine aortic ECs were grown in a collagen gel sandwich to allow tube formation. We found that all three APLs interfered with endothelial tube formation in a dose-dependent manner, with a more than 50% reduction at 25 micromol/l. Interference with the angiogenic process represents a novel mode of action of APLs and might significantly contribute to the antitumor effect of these compounds.

Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts.

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.

Coformulated N-octanoyl-glucosylceramide improves cellular delivery and cytotoxicity of liposomal doxorubicin.

The anticancer agent doxorubicin is in certain cases administered as a long-circulating liposomal formulation. Due to angiogenesis-related structural abnormalities in the endothelial lining of many neoplasms, these complexes tend to extravasate and accumulate in the tumor stroma. However, delivery of doxorubicin is still not optimal since liposomes are not taken up directly by tumor cells. Instead, doxorubicin is gradually released into the interstitial space, and the subsequent uptake by surrounding cells is a limiting step in the delivery process. We recently demonstrated that plasma membrane-inserted short-chain sphingomyelin facilitates the cellular uptake of free doxorubicin. Here, we report that N-octanoyl-glucosylceramide acts equally potent but is itself less toxic. When coformulated with liposomal doxorubicin, this short-chain glycosphingolipid administered to cultured A431 epidermoid carcinoma cells led to superior (up to 4-fold) cellular doxorubicin accumulation and cytotoxicity, compared with control doxorubicin liposomes. These results were fully reproducible when N-octanoyl-glucosylceramide was postinserted into Caelyx, a commercial liposomal doxorubicin preparation. The doxorubicin-potentiating effect of N-octanoyl-glucosylceramide-enriched liposomes proved relatively insensitive to high serum concentrations, indicating that in vivo application is a feasible option. N-Octanoyl-glucosylceramide enrichment might thus represent a major improvement of conventional liposomal doxorubicin formulations.

In vivo imaging of radiation-induced apoptosis in follicular lymphoma patients.

PURPOSE: To evaluate (99m)Tc-Annexin-V (TAV) scintigraphy in monitoring radiation-induced apoptotic cell death in follicular lymphoma (FL) patients. PATIENTS AND METHODS: Eleven FL patients (7 female and 4 male; median age, 58 years; range, 42-80 years) with recurrent disease underwent TAV imaging before and 24 hours after the last fraction of the 2 x 2 Gy involved field radiotherapy regimen. Fine-needle aspiration cytology was performed on 5 consecutive days to determine the optimal time window for apoptosis detection and to confirm the apoptotic nature of the response. The TAV scintigraphy (total body studies and SPECT of the irradiated sites) was performed 4 hours after the administration of the radiopharmaceutical. Tumor uptake was scored in a semiquantitative manner as absent (-) weak (+/-), present (+), or intense (++) with corresponding categories for the cytologic slides. Response evaluation was performed after 1 week and 4 weeks both in terms of completeness and speed of remission. RESULTS: Baseline TAV uptake was absent in 6 and weak in 5 patients. Sequential cytology indicated that the optimal time period for apoptosis assessment was between 24 and 48 hours after the last fraction of the 2 x 2 Gy regimen. Baseline cytology was concordant with baseline TAV in all patients. Apoptotic feature appearance (nuclear chromatin condensation, margination and apoptotic body formation) after low-dose irradiation matched the irradiation response in all patients. In all but 1 patient the posttreatment TAV uptake matched the posttreatment cytology. In these 10 patients the cytology and TAV results correlated with the type and onset of the clinical response. CONCLUSION: Tumor (99m)Tc-Annexin-V uptake can be increased after 2 x 2 Gy involved field radiotherapy. This increase was concordant with the appearance of apoptotic morphology as determined by cytology, and correlated with the clinical outcome. Apoptotic cell death can be observed on Day 4 of this regimen and if so predicts a complete remission within 1 week.

Anti-cancer alkyl-lysophospholipids inhibit the phosphatidylinositol 3-kinase-Akt/PKB survival pathway.

Synthetic alkyl-lysophospholipids (ALPs) represent a new class of anti-tumor agents that target cell membranes and induce apoptosis. However, the exact mechanisms by which ALPs exert these effects remain unclear. Here, we investigated in the epithelial carcinoma cell lines A431 and HeLa the effect of three clinically relevant ALPs [Et-18-OCH3 (Edelfosine), HePC (Miltefosine) and D-21266 (Perifosine)] on the phosphatidylinositol 3-kinase (PI3K)-Akt/PKB survival pathway. We found that growth factor-induced Akt/PKB activation in these cells is dependent on PI3K and that all three ALPs inhibited this pathway in a dose-dependent manner. We further showed that inhibition of the PI3K-Akt/PKB pathway by wortmannin or ALPs is associated with activation of the pro-apoptotic SAPK/JNK pathway. Inhibition of the PI3K-Akt/PKB survival pathway represents a novel mode of action of ALPs that may significantly contribute to the induction of apoptosis.

Submicromolar doses of alkyl-lysophospholipids induce rapid internalization, but not activation, of epidermal growth factor receptor and concomitant MAPK/ERK activation in A431 cells.

Synthetic ALPs, e.g., Et-18-OCH(3) and HePC, are anticancer agents that accumulate in cell membranes, where they interfere with lipid-mediated signal transduction. We previously reported that ALPs, when added at micromolar concentrations (5-25 microM), inhibit growth factor-induced MAPK/ERK activation and enhance radiation-induced apoptosis. We now show that, at nanomolar doses (10-500 nM), ALPs activate the MAPK/ERK pathway in A431 cells without stimulating cell proliferation. Strikingly, ALPs (500 nM) also trigger rapid clustering and internalization of the EGFR in A431 cells. Tyrphostin AG1478, an EGFR tyrosine kinase inhibitor, blocks ALP-induced MAPK/ERK activation but not EGFR internalization. We found no evidence for ALPs acting via G protein-coupled receptors and/or transactivation of EGFRs, as determined by calcium mobilization, EGFR phosphorylation and Grb2 binding assays. Since ALPs readily intercalate into the plasma membrane, our data suggest that they induce subtle changes in the lipid microenvironment of the EGFR, resulting in clustering and internalization of the EGFR and concomitant MAPK/ERK activation.