Phase II Study to Determine the Antitumor Activity and Safety of Simlukafusp Alfa (FAP-IL2v) Combined with Atezolizumab in Esophageal Cancer.

PURPOSE: In this study, we report the results from the esophageal squamous cell carcinoma (SCC) cohort of a phase II, noncomparative, basket study evaluating the antitumor activity and safety of fibroblast activation protein-IL2 variant (FAP-IL2v) plus atezolizumab in patients with advanced/metastatic solid tumors (NCT03386721). PATIENTS AND METHODS: Eligible patients had an Eastern Cooperative Oncology Group performance status of 0 to 1; measurable metastatic, persistent, or recurrent esophageal SCC; progression on ≥1 prior therapy; and were checkpoint inhibitor-naïve. Patients received FAP-IL2v 10 mg plus atezolizumab 1,200 mg intravenously every 3 weeks, or FAP-IL2v weekly for 4 weeks and then every 2 weeks plus atezolizumab 840 mg intravenously every 2 weeks. The primary endpoint was investigator-assessed objective response rate (ORR). RESULTS: In the response-evaluable population (N = 34), the best confirmed ORR was 20.6% [95% confidence interval (CI), 10.4-36.8], with a complete response seen in 1 patient and partial responses in 6 patients. The disease control rate was 44.1% (complete response = 2.9%; partial response = 17.6%; stable disease = 23.5%), and the median duration of response was 10.1 mon/ths (95% CI, 5.6-26.7). The median progression-free survival was 1.9 months (95% CI, 1.8-3.7). Analysis of response by PDL1 expression (Ventana SP263) resulted in an ORR of 26.7% for patients with PDL1-positive tumors (tumor area positivity cutoff ≥1%; n = 15) and 7.1% for patients with PDL1-negative tumors (tumor area positivity cutoff <1%; n = 14). Overall, the treatment combination was tolerable, and adverse events were consistent with the known safety profiles of each drug. CONCLUSIONS: FAP-IL2v plus atezolizumab demonstrated clinical activity and was tolerable in patients with previously treated esophageal SCC.

Characterization and drug resistance patterns of Ewing's sarcoma family tumor cell lines.

Despite intensive treatment with chemotherapy, radiotherapy and surgery, over 70% of patients with metastatic Ewing's Sarcoma Family of Tumors (EFT) will die of their disease. We hypothesize that properly characterized laboratory models reflecting the drug resistance of clinical tumors will facilitate the application of new therapeutic agents to EFT. To determine resistance patterns, we studied newly established EFT cell lines derived from different points in therapy: two established at diagnosis (CHLA-9, CHLA-32), two after chemotherapy and progressive disease (CHLA-10, CHLA-25), and two at relapse after myeloablative therapy and autologous bone marrow transplantation (post-ABMT) (CHLA-258, COG-E-352). The new lines were compared to widely studied EFT lines TC-71, TC-32, SK-N-MC, and A-673. These lines were extensively characterized with regard to identity (short tandem repeat (STR) analysis), p53, p16/14 status, and EWS/ETS breakpoint and target gene expression profile. The DIMSCAN cytotoxicity assay was used to assess in vitro drug sensitivity to standard chemotherapy agents. No association was found between drug resistance and the expression of EWS/ETS regulated genes in the EFT cell lines. No consistent association was observed between drug sensitivity and p53 functionality or between drug sensitivity and p16/14 functionality across the cell lines. Exposure to chemotherapy prior to cell line initiation correlated with drug resistance of EFT cell lines in 5/8 tested agents at clinically achievable concentrations (CAC) or the lower tested concentration (LTC): (cyclophosphamide (as 4-HC) and doxorubicin at CAC, etoposide, irinotecan (as SN-38) and melphalan at LTC; P<0.1 for one agent, and P<0.05 for four agents. This panel of well-characterized drug-sensitive and drug-resistant cell lines will facilitate in vitro preclinical testing of new agents for EFT.

National Cancer Institute pediatric preclinical testing program: model description for in vitro cytotoxicity testing.

BACKGROUND: The National Cancer Institute (NCI) has established the Pediatric Preclinical Testing Program (PPTP) for testing drugs against in vitro and in vivo childhood cancer models to aid in the prioritization of drugs considered for early phase pediatric clinical trials. PROCEDURES: In vitro cytotoxicity testing employs a semi-automated fluorescence-based digital imaging cytotoxicity assay (DIMSCAN) that has a 4-log dynamic range of detection. Curve fitting of the fractional survival data of the cell lines in response to various concentrations of the agents was used to calculate relative IC(50) , absolute IC(50) , and Y(min) values. The panel of 23 pediatric cancer cell lines included leukemia (n = 6), lymphoma (n = 2), rhabdomyosarcoma (n = 4), brain tumors (n = 3), Ewing family of tumors (EFT, n = 4), and neuroblastoma (n = 4). The doubling times obtained using DIMSCAN were incorporated into data analyses to estimate the relationship between input cell numbers and final cell number. RESULTS: We report in vitro activity data for three drugs (vincristine, melphalan, and etoposide) that are commonly used for pediatric cancer and for the mTOR inhibitor rapamycin, an agent that is currently under preclinical investigation for cancer. To date, the PPTP has completed in vitro testing of 39 investigational and approved agents for single drug activity and two investigational agents in combination with various "standard" chemotherapy drugs. CONCLUSIONS: This robust in vitro cytotoxicity testing system for pediatric cancers will enable comparisons to response data for novel agents obtained from xenograft studies and from clinical trials.

Combination of vorinostat and flavopiridol is selectively cytotoxic to multidrug-resistant neuroblastoma cell lines with mutant TP53.

As p53 loss of function (LOF) confers high-level drug resistance in neuroblastoma, p53-independent therapies might have superior activity in recurrent neuroblastoma. We tested the activity of vorinostat, a histone deacetylase inhibitor, and flavopiridol, a pan-Cdk inhibitor, in a panel of multidrug-resistant neuroblastoma cell lines that included lines with wild-type (wt) and transcriptionally active TP53 (n = 3), mutated (mt), and LOF TP53 (n = 4) or p14(ARF) deletion (n = 1). The combination of vorinostat and flavopiridol was synergistic and significantly more cytotoxic (P < 0.001) in cell lines with p53-LOF and in the clones stably transfected with dominant-negative p53 plasmids. Cell cycle analysis by flow cytometry showed prominent cell-cycle arrest in G(2)/M (37%) for a cell line with wt TP53 (SK-N-RA) at 16 to 20 hours, while cells with mt TP53 (CHLA-90) slipped into sub-G(1) at 6 to 24 hours (25%-40% specific cell death). The morphological hallmarks of mitotic cell death, including defective spindle formation and abnormal cytokinesis, were detected by confocal microscopy after the treatment with vorinostat + flavopiridol combination in CHLA-90. The combination caused reduction in the expression of G(2)/M proteins (cyclin B1, Mad2, MPM2) in 2 cell lines with mt TP53 but not in those with wt TP53. Plk1 expression was reduced in all treated lines. Small interfering RNA knockdown of Mad2 and cyclin B1 or Plk1 synergistically reduced the clonogenicity of CHLA-90 cells. The combination of HDAC inhibitor and flavopiridol may be a unique approach to treating neuroblastomas with p53 LOF, one that evokes induction of mitotic failure.

Initial testing (stage 1) of vorinostat (SAHA) by the pediatric preclinical testing program.

Vorinostat, a histone deacetylase inhibitor, was evaluated against the in vitro and in vivo childhood solid tumor and leukemia models in the Pediatric Preclinical Testing Program (PPTP). In vitro testing was performed by the DIMSCAN cytotoxicity assay. In vivo, vorinostat was administered intraperitoneally to mice bearing xenografts. Vorinostat demonstrated 2-log cell growth inhibitory activity in vitro, but generally at concentrations not sustainable in the clinic. No objective responses were observed for any of the solid tumor or acute lymphoblastic leukemia xenografts. Preclinical studies with appropriate drug combinations may provide direction for further clinical evaluations of vorinostat against selected pediatric cancers.

Interleukin-6 in the bone marrow microenvironment promotes the growth and survival of neuroblastoma cells.

Neuroblastoma, the second most common solid tumor in children, frequently metastasizes to the bone marrow and the bone. Neuroblastoma cells present in the bone marrow stimulate the expression of interleukin-6 (IL-6) by bone marrow stromal cells (BMSC) to activate osteoclasts. Here we have examined whether stromal-derived IL-6 also has a paracrine effect on neuroblastoma cells. An analysis of the expression of IL-6 and its receptor, IL-6R, in 11 neuroblastoma cell lines indicated the expression of IL-6 in 4 cell lines and of IL-6R in 9 cell lines. Treatment of IL-6R-positive cells with recombinant human IL-6 resulted in signal transducer and activator of transcription-3 and extracellular signal-regulated kinase-1/2 activation. Culturing IL-6R-positive neuroblastoma cells in the presence of BMSC or recombinant human IL-6 increased proliferation and protected tumor cells from etoposide-induced apoptosis, whereas it had no effect on IL-6R-negative tumor cells. In vivo, neuroblastoma tumors grew faster in the presence of a paracrine source of IL-6. IL-6 induced the expression of cyclooxygenase-2 in neuroblastoma cells with concomitant release of prostaglandin-E2, which increased the expression of IL-6 by BMSC. Supporting a role for stromal-derived IL-6 in patients with neuroblastoma bone metastasis, we observed elevated levels of IL-6 in the serum and bone marrow of 16 patients with neuroblastoma bone metastasis and in BMSC derived from these patients. Altogether, the data indicate that stromal-derived IL-6 contributes to the formation of a bone marrow microenvironment favorable to the progression of metastatic neuroblastoma.

Cdc6 knockdown inhibits human neuroblastoma cell proliferation.

The cell division controller Cdc6 plays a central role in the initiation of DNA replication. It was found that elevated levels of Cdc6 were present in many of human cancer cells, and the accumulation of Cdc6 is required for cell proliferation. In this study, we have investigated the control of Cdc6 expression and its effect on cell proliferation and death in human neuroblastoma cells. Elevated levels of Cdc6 are found in the LA-N-2, CHLA255, and other cell lines that grow fast. Cdc6 knockdown via a Cdc6 short hairpin RNA lentivirus causes the accumulation of sub-G1 populations with the decrease of S contents in the LA-N-2 and CHLA255 cells. Expression profile from the selected genes shows the reduction of cyclin E, cyclin A, and Cdc25C, with a boosted increase of the CDK inhibitor p27Kip1, indicating the suppression of tumor cell proliferation. Further, Cdc6 knockdown causes the increase of pro-apoptotic Bax accompanied with the decrease of anti-apoptotic Bcl-2, resulting in the increased cell death. Furthermore, Cdc6 knockdown causes a sharp reduction of tumor suppressor protein p53, and Cdc6 overexpression renders a boosted p53 expression; and this regulation is at p53 posttranscriptional level. Our study indicates that human Cdc6 functions in several pathways to control the cell proliferation and the cell death.

Initial testing (stage 1) of the proteasome inhibitor bortezomib by the pediatric preclinical testing program.

BACKGROUND: Bortezomib is a proteasome inhibitor that has been approved by FDA for the treatment of multiple myeloma and that has completed phase 1 testing in children. The purpose of the current study was to evaluate the antitumor activity of bortezomib against the in vitro and in vivo childhood cancer preclinical models of the Pediatric Preclinical Testing Program (PPTP). PROCEDURES: Bortezomib was tested against the PPTP in vitro panel at concentrations ranging from 0.1 nM to 1.0 microM and was tested in vivo at a dose of 1 mg/kg for a planned duration of 6 weeks. RESULTS: Bortezomib was uniformly active against the PPTP's in vitro panel, with a median IC(50) of 23 nM and with a steep dose-response curve. The four acute lymphoblastic leukemia (ALL) cell lines had significantly lower IC(50) values compared to the remaining lines of the in vitro panel. Limited in vivo activity was observed for bortezomib against the solid tumor xenografts tested, with one line meeting criteria for intermediate activity for the time to event measure and with the remaining lines showing low activity for this measure. Bortezomib demonstrated in vivo activity against the ALL panel, inducing two complete and two partial responses among seven evaluable lines. CONCLUSIONS: Administered at its MTD in mice, bortezomib demonstrated activity against selected lines of the PPTP's ALL in vivo panel. Further studies are indicated to determine the activity of bortezomib when combined with standard agents to treat childhood ALL.

Flow cytometry analysis of single-strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody.

The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC)+/-pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation+/-hydrogen peroxide (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs.

Histone deacetylase 1 gene expression and sensitization of multidrug-resistant neuroblastoma cell lines to cytotoxic agents by depsipeptide.

BACKGROUND: Genes that are overexpressed in multidrug-resistant neuroblastomas relative to drug-sensitive neuroblastomas may provide targets for modulating drug resistance. METHODS: We used microarrays to compare the gene expression profile of two drug-sensitive neuroblastoma cell lines with that of three multidrug-resistant neuroblastoma cell lines. RNA expression of selected overexpressed genes was quantified in 17 neuroblastoma cell lines by reverse transcription-polymerase chain reaction (RT-PCR). Small-interfering RNAs (siRNAs) were used for silencing gene expression. Cytotoxicity of melphalan, carboplatin, etoposide, and vincristine and cytotoxic synergy (expressed as combination index calculated by CalcuSyn software, where combination index < 1 indicates synergy and > 1 indicates antagonism) were measured in cell lines with a fluorescence-based assay of cell viability. All statistical tests were two-sided. RESULTS: A total of 94 genes were overexpressed in the multidrug-resistant cell lines relative to the drug-sensitive cell lines. Nine genes were selected for RT-PCR analysis, of which four displayed higher mRNA expression in the multidrug-resistant lines than in the drug-sensitive lines: histone deacetylase 1 (HDAC1; 2.3-fold difference, 95% confidence interval [CI] = 1.0-fold to 3.5-fold, P = .025), nuclear transport factor 2-like export factor (4.2-fold difference, 95% CI = 1.7-fold to 7.6-fold, P = .0018), heat shock 27-kDa protein 1 (2.5-fold difference, 95% CI = 1.0-fold to 87.7-fold, P = .028), and TAF12 RNA polymerase II, TATA box-binding protein-associated factor, 20 kDa (2.2-fold, 95% CI = 0.9-fold to 6.0-fold, P = .051). siRNA knockdown of HDAC1 gene expression sensitized CHLA-136 neuroblastoma cells to etoposide up to fivefold relative to the parental cell line or scrambled siRNA-transfected cells (P<.001). Cytotoxicity of the histone deacetylase inhibitor depsipeptide was tested in combination with melphalan, carboplatin, etoposide, or vincristine in five multidrug-resistant neuroblastoma cell lines, and synergistic cytotoxicity was demonstrated at a 90% cell kill of treated cells (combination index < 0.8) in all cell lines. CONCLUSION: High HDAC1 mRNA expression was associated with multidrug resistance in neuroblastoma cell lines, and inhibition of HDAC1 expression or activity enhanced the cytotoxicity of chemotherapeutic drugs in multidrug-resistant neuroblastoma cell lines. Thus, HDAC1 is a potential therapeutic target in multidrug-resistant neuroblastoma.

Assessing combinations of cytotoxic agents using leukemia cell lines.

The mainstay of clinical anti-neoplastic chemotherapy is multi-agent combinations, most of which were developed empirically. To speed research and decrease costs, there is increasing interest in moving new drugs into clinical trials in potentially active combinations based upon pre-clinical testing data. Because testing drug combinations in animals is expensive and complex, defining drug combinations initially in cell culture assays is essential. For in vitro testing we employ a panel of well-characterized cell lines and DIMSCAN, a semi-automatic fluorescence-based digital image microscopy system that quantifies relative total (using a DNA stain) or viable [using fluorescein diacetate (FDA)] cell numbers in tissue culture multi-well-plates ranging from 6 to 384 wells per plate. DIMSCAN is a rapid and efficient tool for conducting in vitro cytotoxicity assays across a 4 log dynamic range. The specificity of detecting viable cells with FDA is achieved by use of digital image processing and chemical quenching of fluorescence in non-viable cells with eosin Y. Cytotoxicity measured by DIMSCAN was found to be comparable to manual trypan blue dye exclusion counts or colony formation in soft agar, but with a significantly wider dynamic range, that enables drug combination studies used to detect synergistic or antagonistic interactions in cell lines from both solid tumors and leukemias. While different mathematical models have been proposed for evaluating drug interactions, which can be classified as synergistic (combinations demonstrating greater than the additive activity expected from each agent alone), additive, or antagonistic (drugs showing less activity in combination than expected from the sum of each agent alone), we generally find the Combination Index method (as developed by Chou, et al.) to be the most suitable for evaluating of drug interactions in cell culture assays.

E-cadherin cell-cell adhesion in ewing tumor cells mediates suppression of anoikis through activation of the ErbB4 tyrosine kinase.

Ability to grow under anchorage-independent conditions is one of the major hallmarks of transformed cells. Key to this is the capacity of cells to suppress anoikis, or programmed cell death induced by detachment from the extracellular matrix. To model this phenomenon in vitro, we plated Ewing tumor cells under anchorage-independent conditions by transferring them to dishes coated with agar to prevent attachment to underlying plastic. This resulted in marked up-regulation of E-cadherin and rapid formation of multicellular spheroids in suspension. Addition of calcium chelators, antibodies to E-cadherin (but not to other cadherins or beta(1)-integrin), or expression of dominant negative E-cadherin led to massive apoptosis of spheroid cultures whereas adherent cultures were unaffected. This correlated with reduced activation of the phosphatidylinositol 3-kinase-Akt pathway but not the Ras-extracellular signal-regulated kinase 1/2 cascade. Furthermore, spheroid cultures showed profound chemoresistance to multiple cytotoxic agents compared with adherent cultures, which could be reversed by alpha-E-cadherin antibodies or dominant negative E-cadherin. In a screen for potential downstream effectors of spheroid cell survival, we detected E-cadherin-dependent activation of the ErbB4 receptor tyrosine kinase but not of other ErbB family members. Reduction of ErbB4 levels by RNA interference blocked Akt activation and spheroid cell survival and restored chemosensitivity to Ewing sarcoma spheroids. Our results indicate that anchorage-independent Ewing sarcoma cells suppress anoikis through a pathway involving E-cadherin cell-cell adhesion, which leads to ErbB4 activation of the phosphatidylinositol 3-kinase-Akt pathway, and that this is associated with increased resistance of cells to cytotoxic agents.

In vitro testing of chemosensitivity in physiological hypoxia.

Highly aggressive, rapidly growing tumors are often hypoxic, owing to an inadequate supply relative to consumption of oxygen (O2) in the expanding tumor mass, or growth in tissues with physiologically low O2 concentrations (such as bone marrow). Selection of tumor cells that can grow or survive under hypoxic conditions appears from both experimental and clinical studies to impact tumor progression, response to therapy, and to increase resistance to radiation and to certain cytotoxic drugs. Therefore, the predictive value of preclinical testing of anticancer agents in cell culture might be improved by conducting testing in conditions of physiological hypoxia. We review the impact of hypoxia on anticancer drug cytotoxicity and the methods used in our laboratory to asses the cytotoxic activity of single antineoplastic drugs under conditions of physiological hypoxia.

DIMSCAN: a microcomputer fluorescence-based cytotoxicity assay for preclinical testing of combination chemotherapy.

DIMSCAN is a semiautomatic fluorescence-based digital image microscopy system that quantifies relative total (using a DNA stain) or viable (using fluorescein diacetate [FDA]) cell numbers in tissue culture multiwell plates ranging from 6 to 384 wells per plate. DIMSCAN is a rapid and efficient tool for conducting in vitro cytotoxicity assays across a 4 log dynamic range. The specificity of detecting viable cells with FDA is achieved by using digital image processing and chemical quenching of fluorescence in nonviable cells with eosin Y. Average scan time for the most commonly used format, a 96-well plate, is 6 min. Cytotoxicity for neuroblastoma cell lines measured by DIMSCAN was found to be comparable to manual Trypan blue dye exclusion counts or colony formation in soft agar, but with a significantly wider dynamic range, which enables drug combination studies used to detect synergistic or antagonistic interactions. The linearity of DIMSCAN was validated (r2 = 0.99967 +/- 0.0003) for cells stained with FDA deposited using a fluorescence-activated cell sorter, documenting a dynamic range > 4 logs, and the ability to detect a single viable cell in a well 93% of the time. DIMSCAN has been used to demonstrate preclinical activity of cytotostatic and cytotoxic drugs and drug combinations that have subsequently shown activity in clinical trials.

Pyrazoloacridine is active in multidrug-resistant neuroblastoma cell lines with nonfunctional p53.

PURPOSE: The purpose of this study was to determine the activity of pyrazoloacridine (PZA) in neuroblastomas that have acquired high-level resistance to multiple drugs (not associated with multidrug resistance-associated protein or P-glycoprotein) during therapy, including those with loss of p53 function. EXPERIMENTAL DESIGN: We determined the activity of PZA in 12 drug-sensitive and 10 multidrug-resistant (MDR) neuroblastoma cell lines. Six of the MDR cell lines lacked functional p53. Drug cytotoxicity was measured using the DIMSCAN fluorescence/digital imaging microscopy assay with a 4-log dynamic range. RESULTS: LC(90) (i.e., the drug concentration that was lethal for 90% of the cell population) values ranged from 0.01 to 1.1 microM for the drug-sensitive cell lines, from 0.8 to 2.4 microM for the MDR cell lines with functional p53, and from 0.9 to 2.1 microM for the MDR cell lines that lacked functional p53. To confirm that PZA cytotoxicity is independent of p53 function, we compared two neuroblastoma cell lines in which p53 function was abrogated via human papilloma virus-16 E6 transduction (which mediates increased degradation of p53) to the mock-transduced (LXSN) controls. LC(90) values for human papilloma virus-16 E6 clones (abrogated p53) ranged from 0.2 to 2.04 micro M, whereas LC(90) values for LXSN controls (functional p53) were 0.1 and 0.5 microM. PZA was active with 72-h in vitro exposures against p53-nonfunctional MDR cells at drug levels (2-3 microM) obtained for shorter periods (1-3 h) in Phase I and II clinical trials. Washout experiments showed that 3 micro M PZA achieved 0.5-1 log of cell kill with 1-3-h exposures versus 3 logs at 24 h. CONCLUSIONS: These data suggest that expanding the time of PZA systemic exposure, which may be clinically tolerable with hematopoietic stem cell support, should be tested in clinical trials. Prolonged systemic exposure to PZA with hematopoietic stem cell support may be effective against recurrent neuroblastomas that have failed conventional chemotherapeutic regimens, including those neuroblastomas with loss of p53 function.

Antagonism of buthionine sulfoximine cytotoxicity for human neuroblastoma cell lines by hypoxia is reversed by the bioreductive agent tirapazamine.

Relapse of neuroblastoma (NB) commonly occurs in hypoxic tissues. Buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, is cytotoxic for NB cell lines in atmospheric oxygen (20% O(2)). Tirapazamine (TPZ) is a bioreductive agent that forms a toxic-free radical in hypoxia. We determined in four NB cell lines cytotoxicity using the DIMSCAN digital imaging fluorescence assay, glutathione (GSH) levels by the DTNB-GSSG reductase method, apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (Delta psi(m)) by flow cytometry. Hypoxia (2% O(2)) antagonized BSO-mediated ROS, apoptosis, and cytotoxicity but not GSH depletion. TPZ synergistically enhanced BSO cytotoxicity in hypoxia for all four NB cell lines, achieving 2-4 logs of cell kill. BSO depleted GSH (8-42% of controls) in 20 and 2% O(2), whereas TPZ only decreased GSH in hypoxia. Maximal GSH depletion was induced by BSO + TPZ. N-acetylcysteine abrogated GSH depletion caused by TPZ but not by BSO. BSO increased ROS, decreased Delta psi(m), and caused apoptosis in 20% O(2) (but not in 2% O(2)). TPZ elevated ROS in 2% O(2) (but not in 20% O(2)), whereas BSO + TPZ increased ROS both in 20 and 2% O(2). In hypoxia, TPZ alone or TPZ + BSO caused an 80% decrease of Delta psi(m) at 24 h, preceding apoptosis in 74-86% of cells at 48 h. Thus, hypoxia significantly antagonizes BSO-mediated cytotoxicity for NB cell lines, but TPZ reversed the inhibition of BSO-mediated cytotoxicity in hypoxia, causing increased ROS, Delta psi(m) decrease, GSH depletion, apoptosis, and synergistic cytotoxicity. These data additionally define the role of ROS in BSO-mediated cytotoxicity and suggest that combining BSO with TPZ could have clinical activity against NB in hypoxic sites.