Robert M. Sutphin

Prognostic significance of early lymphocyte recovery in pediatric osteosarcoma

Evidence suggests early lymphocyte recovery after chemotherapy predicts superior outcome for patients with cancer, a phenomenon not previously investigated in osteosarcoma. This study determined the prognostic significance of early lymphocyte recovery for pediatric patients with osteosarcoma.

Mifamurtide in Metastatic and Recurrent Osteosarcoma: A Patient Access Study with Pharmacokinetic, Pharmacodynamic, and Safety Assessments

This non‐randomized, patient‐access protocol, assessed both safety and efficacy outcomes following liposomal muramyl‐tripeptide‐phosphatidylethanolamine (L‐MTP‐PE; mifamurtide) in patients with high‐risk, recurrent and/or metastatic osteosarcoma.

Tolfenamic acid inhibits neuroblastoma cell proliferation and induces apoptosis: A novel therapeutic agent for neuroblastoma

Current therapeutic options for recurrent neuroblastoma have poor outcomes that warrant the development of novel therapeutic strategies. Specificity protein (Sp) transcription factors regulate several genes involved in cell proliferation, survival, and angiogenesis. Sp1 regulates genes believed to be important determinants of the biological behavior of neuroblastoma. Tolfenamic acid (TA), a non‐steroidal anti‐inflammatory drug, is known to induce the degradation of Sp proteins and may serve as a novel anti‐cancer agent. The objective of this investigation was to examine the anti‐cancer activity of TA using established human neuroblastoma cell lines. We tested the anti‐proliferative effect of TA using SH‐SY5Y, CHLA90, LA1 55n, SHEP, Be2c, CMP 13Y, and SMS KCNR cell lines. Cells were treated with TA (0/25/50/100 µM) and cell viability was measured at 24, 48, and 72 h post‐treatment. Selected neuroblastoma cell lines were treated with 50 µM TA for 24 and 48 h and tested for cell apoptosis using Annexin‐V staining. Caspase activity was measured with caspase 3/7 Glo kit. Cell lysates were prepared and the expression of Sp1, survivin, and c‐PARP were evaluated through Western blot analysis. TA significantly inhibited the growth of neuroblastoma cells in a dose/time‐dependent manner and significantly decreased Sp1 and survivin expression. Apart from cell cycle (G0/G1) arrest, TA caused significant increase in the apoptotic cell population, caspase 3/7 activity, and c‐PARP expression. These results show that TA effectively inhibits neuroblastoma cell growth potentially through suppressing mitosis, Sp1, and survivin expression, and inducing apoptosis. These results show TA as a novel therapeutic agent for neuroblastoma.

Abstract 5431: Tolfenamic acid enhances the therapeutic efficacy of certain chemotherapeutic agents in medulloblastoma cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tolfenamic acid (TA), a small molecule NSAID exhibits anti-cancer response in several malignancies. Recently we demonstrated for the first time that TA inhibits medulloblastoma (MB) cell proliferation and tumor growth in mice xenografts. Since MB requires intensive therapy that often causes long-term side-effects, we tested the efficacy of TA for enhancing the response of chemotherapy. Initial experiments were performed using MB cell lines, DAOY and D283 to test the anti-proliferative response of individual agents, TA, Irinotecan (IRI), Topotecan (TOPO), Temozolomide (TZM) and Doxorubicin (DOXO). Subsequently, the combination of TA with TOPO was tested using the optimized doses. DAOY and D283 cells were treated with DMSO or increasing concentrations of TA (5-50 μg/ml) or chemotherapeutic agents TOPO (10-500 nM), TZM (100-500 μM), IRI (0.5-5 μM) and DOXO (10-500 nM). Cell viability was measured at 24, 48, and 72 h post-treatment using CellTiter Glo kit and all agents caused a dose and time-dependent inhibition of cell viability. To assess the effect of combination therapy, MB cells were treated with optimized doses of TA (10 μg/ml) or TOPO (DAOY: 20 nM; D283: 25 nM) or both and the cell viability was measured at 48 h and 72 h post-treatment. The combination of TA and TOPO caused significantly higher inhibition when compared to either TA or TOPO alone confirming the efficacy of this combination therapy in pre-clinical models for MB. The effect on cell apoptosis was evaluated at 48 h post-treatment. Apoptotic cells were measured by flow cytometry using Annexin V-PE/7-AAD kit. The results showed a significant increase in the apoptotic fraction (annexin V positive) of both MB cell lines following the combination therapy when compared to individual treatment of TA or TOPO. These results were further supported by determining the activity of effector caspases. Consistent with Annexin V staining, combination therapy significantly upregulated caspase 3/7 levels when compared to individual treatment. DAOY and D283 were treated with DMSO or 10 or 20 µg/ml TA for 48 h. Whole cell lysates were prepared and the expression of Sp1, survivin and c-PARP was determined by Western blot analysis. TA inhibited both Sp1 and survivin in MB cell lines. Consistent with annexin V staining and caspase 3/7 results, TA augmented the PARP cleavage confirming the activation of apoptotic pathways. In order to evaluate the precise molecular markers/pathways associated with the beneficial (higher) efficacy of TA and TOPO combination therapy, we are conducting molecular profiling analysis. Overall, these results demonstrate that the combination of anti-cancer small molecule, TA and standard chemotherapeutic agents such as TOPO effectively inhibits MB cell growth. These preliminary results strongly demonstrate the efficacy of the proposed combination therapy in enhancing therapeutic response in pre-clinical models of MB. Citation Format: Don Eslin, Umesh T. Sankpal, Chris M. Lee, Robert M. Sutphin, W. Paul Bowman, Jeffrey C. Murray, Riyaz Basha. Tolfenamic acid enhances the therapeutic efficacy of certain chemotherapeutic agents in medulloblastoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5431. doi:10.1158/1538-7445.AM2014-5431

Abstract 3382: Nifurtimox enhances the therapeutic efficacy of radiation by inducing reactive oxygen species and related pathways: Pre-clinical study in medulloblastoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for about 20% of pediatric CNS tumors. Multimodality treatments including surgery, radiotherapy (XRT) and chemotherapy have greatly improved disease outcome but survivors often suffer from deleterious effects of aggressive treatment with XRT. The aim of this study is to use novel strategies to enhance the response of XRT in a pre-clinical model for MB. XRT is known to cause DNA damage and cell death by inducing reactive oxygen species (ROS). Induction of ROS causes perturbation in antioxidant defense system thereby causing cell damage and death. Therefore, induction of ROS may serve as a promising strategy in cancer therapy. Nifurtimox (Nfx), a nitrofuran compound used to treat the parasitic infection Chagas’ disease, is known to induce ROS in pre-clinical models for neuroblastoma (NB) and MB. It is currently in clinical trials for treating NB and MB in children. In this study, we investigated the effects of Nfx in augmenting the efficacy of XRT in MB cell lines. DAOY and D283 cells were treated with varying dose of Nfx (5-70 µg/ml) or XRT (2-10 Gy) and cell viability was assessed using CellTiter-Glo for up to 5 days. The effect of combination therapy on cell viability, apoptosis, ROS levels and catalase activity was evaluated using optimized dose of Nfx and XRT. Results revealed that combination treatment significantly inhibited MB cell growth when compared to the effect of either Nfx or XRT alone. This was accompanied by the activation of apoptotic cell death as determined by increased annexin V staining and caspase 3/7 activity. An increase in ROS was also observed following combination treatment with Nfx and XRT which correlated with cell growth inhibition. Interestingly, catalase activity was unaltered following the treatment with single or double agents suggesting a contribution of impaired antioxidant system in causing MB cell growth inhibition. To further delineate the underlying mechanisms and to identify other candidates modulated by this combination therapy, we undertook molecular profiling approach using Affimetrix gene expression arrays (Human Gene 2.0 ST Array). We examined differentially expressed genes and affiliated pathways, and biological processes underlying tumor cell responses to Nfx and XRT. Our results identified genes that are statistically enriched for biological functions related to tumorigenesis, including cancer cell death and survival, cell cycle, and other functions relating to cancer disease. Several transcription factors and their downstream targets were also found to be enriched including the NRF2-mediated oxidative stress response pathway that responds to various stimuli including ROS. In conclusion, this pre-clinical study demonstrates that Nfx has the potential to enhance the therapeutic efficacy of radiation in MB cell lines. Citation Format: Don Eslin, Umesh T. Sankpal, Chris M. Lee, Giselle Saulnier Sholler, Robert M. Sutphin, Paul Bowman, Jeffrey C. Murray, Riyaz M. Basha. Nifurtimox enhances the therapeutic efficacy of radiation by inducing reactive oxygen species and related pathways: Pre-clinical study in medulloblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3382. doi:10.1158/1538-7445.AM2014-3382

Abstract 2770: Anti-leukemic response of a NSAID, tolfenamic acid.

Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug is known to inhibit human cancer cells and mouse tumor growth in some cancer models; however its anti-leukemic response is not yet evaluated. Research from our laboratory and others showed that TA targets specificity protein (Sp) transcription factors which mediate the expression of several genes associated with cancer. We also showed that TA inhibits the expression of survivin, a key member of inhibitor of apoptosis family in several human cancer cells. Recent work from several laboratories revealed a strong association of survivin in leukemia. We hypothesize by targeting Sp proteins and survivin, TA can act as an anti-leukemic agent. The anti-proliferative response of TA was determined using four human leukemia cell lines, Jurkat (acute T-cell leukemia), Nalm-6 (pre-B cell leukemia), Molt-4 (acute lymphoblastic leukemia; T lymphoblast), and Reh (acute lymphoblastic leukemia, non-T; non-B) Cells were treated with increasing (25/50/75/100 μM) concentrations of TA and the cell viability was measured at 24, 48, and 72 h post-treatment using CellTiter-Glo kit. Results show a consistent decrease in cell viability in a dose and time-dependent manner. Confirmatory studies to elucidate the mechanism of action were conducted using selected cell lines, Jurkat and Nalm-6. Apoptosis and cell cycle analysis was performed using flow cytometry. The expression of c-PARP, Sp1, survivin, CDC2, CDC4, Cyclin D3 and pRb was determined by Western blot analysis and the caspases were measured by using Caspse-Glo kit(s). Results showed a significant increase in the apoptotic (annexin V positive) cell population following TA treatment, while cell cycle phase distribution analysis showed G 0 /G 1 arrest. TA-induced cell apoptosis is supported by robust activation of caspases (3/7, 8 and 9), and the expression of c-PARP. TA down-regulated the expression of CDC2, CDC4, Cyclin D3 and pRb that mediate the early phases of cell cycle. In summary, TA modulated the expression of critical candidate genes associated with the early phases of cell cycle with validated efficacy in causing G 0 /G 1 arrest. Western blot results reveal that TA significantly decreases Sp1 and survivin expression. Further work is needed to clarify the role of TA as a novel therapeutic agent for leukemia. Citation Format: Robert M. Sutphin, Sarah F. Connelly, Chris M. Lee, Umesh T. Sankpal, Don Eslin, Riyaz Basha. Anti-leukemic response of a NSAID, tolfenamic acid. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2770. doi:10.1158/1538-7445.AM2013-2770

Abstract 2767: Nifurtimox and radiation for treating neuroblastoma: a preclinical study.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Neuroblastoma (NB) is the most common extracranial solid malignancy in infants and children. NB is responsible for more than 15% of deaths due to childhood cancers. It is an aggressive malignancy and often requires intensive multimodality treatment that potentially causes extensive long term side-effects. Although radiation therapy (XRT) is an important part of the standard treatment plan in this malignancy, XRT is known to cause significant side effects and morbidity associated with XRT greatly impacts the lives of many patients. Therefore, new approaches to increase the efficacy of XRT while decreasing side effects could have a great impact in the use of this therapy in cancer. XRT-induced cell death is partially associated with the generation of reactive oxygen species (ROS). ROS production is implicated in cell apoptosis, and induction of ROS potentially serves as a promising strategy for inhibiting tumor growth. In this investigation we exploited a strategy to increase the response of XRT by adding a chemical agent, Nifurtimox (Nfx) that is known to induce the generation of ROS. Human NB cells, SH-SY5Y and LA1-55n were treated with vehicle (DMSO) or increasing (5/7.5/10/20 μl/ml) concentrations of Nfx or XRT (1/2/5/10 Gy) and cell viability was determined at 24, 48 and 72 hr post-treatment. Both Nfx and XRT inhibited NB cell growth following a dose and time-dependent response. In order to test the combination response, these cells were treated with selected doses of Nfx and/or XRT and the cell viability was measured at 48 h post-treatment. The combination of both agents resulted in significantly higher inhibition when compared to single agent. ROS levels and apoptosis were assessed in NB cells following individual and combination treatment. ROS levels were measured using flow cytometry. Activation of caspases was determined by Caspase Glo kits while apoptotic cells were measured by flowcytometry (Annexin-V staining). Consistent with cell viability results, combination of XRT and Nfx significantly up-regulated ROS, caspases and induced apoptotic cell population when compared to individual treatment. For the in vivo assay, athymic nude mice were subcutaneously injected with LA1-55n cells. When tumor growth volume reached 100 mm3, mice were treated with Nfx (25 mg/kg/day in corn oil through oral gavage for 3 wk), XRT (5 Gy 2 time/wk for 2 wk) or both and the tumor growth was measured. At the termination of the experiment, combination therapy (XRT and Nfx) caused more than 80% tumor growth inhibition while the individual treatment (XRT or Nfx) caused approximately 60% inhibition of tumor growth. Results of this investigation demonstrate that the combination of XRT and Nfx synergistically inhibits human neuroblastoma cell proliferation and tumor growth in mice. Citation Format: Don Eslin, Chris M. Lee, Giselle S. Sholler, Umesh T. Sankpal, Robert M. Sutphin, Riyaz M. Basha. Nifurtimox and radiation for treating neuroblastoma: a preclinical study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2767. doi:10.1158/1538-7445.AM2013-2767

Abstract 1807: Tolfenamic acid inhibits medulloblastoma cell proliferation and tumor growth in mice by targeting Sp1 and survivin.

Medulloblastoma (MB) is the most common malignant tumor of the central nervous system in children. MB requires intensive multimodality treatment that often causes extensive long term side-effects. The morbidities associated with intensive therapy for treating this malignancy are a serious concern in children. Hence research focusing on identifying novel agents with less toxicity is very important. Specificity protein1 (Sp1) is a transcription factor which regulates several genes involved in cell proliferation and cell survival. Sp1 is also known to mediate the expression of survivin, a member of the inhibitor of apoptosis protein family that is associated with aggressive disease and poor prognosis in multiple human cancers. Recently we showed that Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug (NSAID) inhibits neuroblastoma cell growth by targeting Sp1 and survivin. Strategies to target Sp1 and transcriptional regulation of survivin for treating MB have not yet been evaluated. The aim of the current investigation was to test the anti-cancer activity of TA using in vitro assays and a mouse model for MB. Human MB cells, DAOY and D283, were treated with vehicle (DMSO) or increasing concentrations (5-20 μg/ml) of TA and cell viability was measured at 24, 48 and 72 hours post-treatment. TA inhibited MB cell growth in a time and dose dependent manner. MB cells were treated with vehicle (DMSO) or TA (10 or 20 μg/ml) and the effect on cell apoptosis was measured. Apoptotic cells were measured by flow cytometry (Annexin-V staining) and caspase (3/7, 8, and 9) activity was determined using Caspase Glo kits. TA up-regulated caspase activity and augmented the apoptotic cell population. The expression of Sp1, c-PARP and survivin was determined by Western blot analysis. TA significantly inhibited the expression of Sp1, c-PARP, and survivin. Cell cycle phase distribution was measured using flow cytometry and results revealed that TA caused G0/G1 arrest in MB cells. For the in vivo assay, athymic nude mice were subcutaneously injected with D283 cells and treated with TA (50 mg/kg 3 times/wk) for 4 weeks. TA treatment caused 30-40% decrease in tumor weight and volume. Tumor tissue was subjected to immunohistochemical analysis to evaluate the expression of Sp1 and survivin. Consistent with in vitro results, TA-induced tumor growth inhibition in mice was accompanied with a decrease in Sp1 and survivin expression in mice tumor tissue. These pre-clinical data demonstrate that TA can act as an anti-cancer agent in medulloblastoma potentially targeting Sp1 and survivin. Citation Format: Don Eslin, Chris M. Lee, Umesh T. Sankpal, Robert M. Sutphin, Riyaz Basha. Tolfenamic acid inhibits medulloblastoma cell proliferation and tumor growth in mice by targeting Sp1 and survivin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1807. doi:10.1158/1538-7445.AM2013-1807

Abstract 277: The small molecule tolfenamic acid inhibits neuroblastoma cell growth through inducing apoptosis and causing cell cycle arrest potentially targeting Sp1 and survivin

Neuroblastoma is an embryonal tumor of the peripheral sympathetic nervous system found in infants and children. Despite intensive therapy, the mortality rate of this malignancy remains more than 50%. The side effects of standard therapy can cause acute and long term damage and toxicity in various biological functions and can lead to the development of secondary tumors. Therefore there is an urgent need in identifying novel strategies in neuroblastoma that improve out come with less toxicity. Specificity proteins (Sp) regulate several important genes associated with neuroblastoma including MYCN and TrkA. Our aim was to exploit the strategy of targeting Sp proteins using TA in the treatment of neuroblastoma. In this study, we examined the anti-proliferative activity of TA using 7 neuroblastoma cell lines and evaluated the ability of TA to induce apoptosis, and cell cycle arrest using selected cell lines. Apoptosis in mammalian cells primarily takes place through extrinsic and intrinsic pathways. Specificity protein (Sp) transcription factors regulate several genes involved in cell proliferation, survival and angiogenesis. Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug, is known to induce the degradation of Sp proteins and may serve as a novel anti-cancer agent. The objective of this investigation was to examine the anti-cancer activity of TA using established human neuroblastoma cell lines. We tested the anti-proliferative effect of TA using 7 human neuroblastoma cell lines. Cells were treated with TA (0/25/50/100μM) and cell viability was measured at 24, 48, and 72 h post-treatment. Selected neuroblastoma cell lines, LA1 55n and SH-SY5Y were treated with 50 μM TA for 24 and 48 h and tested for cell apoptosis using Annexin-V staining. Caspase activity was measured with caspase 3/7 Glo kit. Cell lysates were prepared and the expression of Sp1, survivin and c-PARP were evaluated through Western blot analysis. TA significantly inhibited the growth of neuroblastoma cells in a dose/time-dependent manner and significantly decreased Sp1 and survivin expression. Apart from cell cycle (G0/G1) arrest, TA caused significant increase in the apoptotic cell population, caspase 3/7 activity and c-PARP expression. These results show that TA effectively inhibits neuroblastoma cell growth potentially through suppressing mitosis, Sp1 and survivin expression, and inducing apoptosis. These results represent a novel strategy for using small molecules for the treatment of neuroblastoma.(Financial assistance: This study is supported by MD Anderson Cancer Center Orlando (RB); Run Way to Hope and Hyundai Hope on Wheels Scholar grant awarded to DE). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 277. doi:1538-7445.AM2012-277