Chris M. Lee

Small molecule tolfenamic acid inhibits PC‐3 cell proliferation and invasion in vitro, and tumor growth in orthotopic mouse model for prostate cancer

Specificity protein (Sp) transcription factors are implicated in critical cellular and molecular processes associated with cancer that impact tumor growth and metastasis. The non‐steroidal anti‐inflammatory drug, tolfenamic acid (TA) is known to inhibit Sp proteins in some human cancer cells and laboratory animal models. We evaluated the anti‐cancer activity of TA using in vitro and in vivo models for prostate cancer.

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.

Bioluminescence imaging correlates with tumor progression in an orthotopic mouse model of lung cancer

BACKGROUND AND OBJECTIVES To determine whether bioluminescence imaging of human lung cancer cells growing in an orthotopic murine model provides a sensitive tool for monitoring tumor progression in athymic nude mice. METHODS Human lung cancer (A549) cells were stably transfected with the firefly luciferase gene and inoculated into the right lung of athymic nude mice. Seven days after inoculation tumor growth was evaluated using the Kodak in-vivo Imaging System FX and continued to be monitored on a weekly basis. RESULTS In duplicate experiments, human lung cancer tumors formed in 90% of animals injected orthotopically. The mean intensity of the bioluminescence signal emitted from the lung cancer cells increased logarithmically during the course of study. Mice with positive bioluminescence signaling had confirmed tumors by microscopic histological analysis. Bioluminescence activity had a strong correlation with the tumor volume as determined histologically. CONCLUSIONS Bioluminescence intensity directly correlates with tumor volume and therefore offers a reliable approach for detecting and monitoring the growth of human lung cancer cells in orthotopic murine models.

Cellular and Organismal Toxicity of the Anti-Cancer Small Molecule, Tolfenamic Acid: A Pre-Clinical Evaluation

Background/Aims: The small molecule, Tolfenamic acid (TA) has shown anti-cancer activity in pre-clinical models and is currently in Phase I clinical trials at MD Anderson Cancer Center Orlando. Since specificity and toxicity are major concerns for investigational agents, we tested the effect of TA on specific targets, and assessed the cellular and organismal toxicity representing pre-clinical studies in cancer. Methods: Panc1, L3.6pl, and MiaPaCa-2 (pancreatic cancer), hTERT-HPNE(normal), and differentiated/un-differentiated SH-SY5Y (neuroblastoma) cells were treated with increasing concentrations of TA. Cell viability and effect on specific molecular targets, Sp1 and survivin were determined. Athymic nude mice were treated with vehicle or TA (50mg/kg, 3times/week for 6 weeks) and alterations in the growth pattern, hematocrit, and histopathology of gut, liver, and stomach were monitored. Results: TA treatment decreased cell proliferation and inhibited the expression of Sp1 and survivin in cancer cells while only subtle response was observed in normal (hTERT-HPNE) and differentiated SH-SY5Y cells. Mice studies revealed no effect on body weight and hematocrit. Furthermore, TA regimen did not cause signs of internal-bleeding or damage to vital tissues in mice. Conclusion: These results demonstrate that TA selectively inhibits malignant cell growth acting on specific targets and its chronic treatment did not cause apparent toxicity in nude mice.

Abstract 2159: Expression of Sp1 and survivin in ovarian cancer specimens: potential novel therapeutic targets in disease treatment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Ovarian cancer (OC) is one of the most common female cancers and is the leading cause of death from gynecologic malignancies. Although, cisplatin is the front-line chemotherapeutic option for OC, its success is compromised due to dose-limiting toxicity and acquired resistance by tumor cells. Specificity protein 1 (Sp1) is a transcription factor that is over-expressed in several cancers and is inversely associated with survival. Survivin, a member of IAP family, is known to cause resistance to chemo- and radiation therapy. Studies from our laboratories have shown that the NSAID, tolfenamic acid (TA), targets Sp1 protein and inhibits expression of survivin in various cancer models. Our primary objective was to evaluate the expression of Sp1 and survivin in OC patients’ specimens and target these candidates using TA for enhancing the cisplatin response. Expression of Sp1 and survivin in clinical specimens was determined by qPCR and Western blots. qPCR showed increased expression of survivin (∼5-fold) and Sp1 (∼2-fold) in tumor samples. Western blot also revealed over-expression of both Sp1 (>2.6-fold) and survivin (>100-fold). OC cell lines (ES2, OVCAR-3) were used to determine the anti-proliferative response to cisplatin and TA. TA and cisplatin showed a dose- and time-dependent inhibition of cell viability in OC cell lines [TA (50 ÂμM) caused 50% (ES2) and 40% (OVCAR-3) growth inhibition and cisplatin (5 ÂμM) caused 60% and 40% inhibition at 48 h post-treatment]. Combination treatment using optimized doses of TA (50 ÂμM) and cisplatin (5 ÂμM) resulted in a synergistic response and caused stronger inhibition (ES2: ∼80%, OVCAR-3: >60) compared to single-agent. Increased inhibition of proliferation by the combination of TA and cisplatin was accompanied by cell-cycle arrest, predominantly in the G2/M phase. A significant increase in apoptosis, as determined by Caspase 3/7 activity, annexin-V staining, and PARP cleavage, was also observed in the combination treatment. Cell invasion and migration was assessed using matrigel coated transwell chambers. Compared to TA or cisplatin treatment alone, their combination significantly inhibited ES2 cell invasion. Analysis of ES2 cells by global proteomic profiling indicated that the combination treatment upregulated proteins associated with oxidative phosphorylation, apoptosis, and electron transport chain; and down-regulated cytoplasmic ribosomal proteins, translational factors, and proteins involved in DNA damage response, and cell cycle. In conclusion, elevated expression of Sp1 and survivin confirmed their association in OC and demonstrated the relevance of targeting these candidates, which may render the OC cells more sensitive to chemotherapy and offer therapeutic potential. Further studies to delineate underlying mechanism(s) of action possibly involving signaling pathways and reactive oxygen species are currently underway. Citation Format: Umesh T. Sankpal, Susan B. Ingersoll, Mohammed I. Shukoor, Chris M. Lee, Nicole M. Stavitzski, Vadiraja B. Bhat, Sarfraz Ahmad, Robert W. Holloway, Liz Abraham, Riyaz Basha. Expression of Sp1 and survivin in ovarian cancer specimens: potential novel therapeutic targets in disease treatment. [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 2159. doi:10.1158/1538-7445.AM2013-2159

Abstract B11: Nifurtimox and radiation combination increases reactive oxygen species and induces apoptosis in medulloblastoma cells

Radiation (XRT) is a part of standard therapy for treating medulloblastoma (MB) in children. Morbidities associated with irradiation often cause long-term side-effects. Here we are testing a strategy to enhance the response of radiation using pre-clinical models for MB. XRT-induced cell death is partially associated with the generation of reactive oxygen species (ROS). Since ROS production is implicated in cell apoptosis, induction of increased ROS may serve as a promising strategy for inhibiting cancer cell growth. Nifurtimox (Nfx), a nitrofuran compound used to treat the parasitic infection Chagas9 disease, is known to induce ROS in pre-clinical models for neuroblastoma and medulloblastoma and is currently in clinical trials for treating these malignancies in children. We investigated the efficacy of Nfx in augmenting the effect of XRT using MB cell lines (DAOY and D283) and a patient-derived primary culture (003BN cells). DAOY, D283, and 003BN cells were treated with vehicle (DMSO) or increasing concentrations/doses of Nfx (5-70 µg/ml) or XRT (2-10 Gy) and cell viability was measured up to 3 (MB cell lines) or 5 (primary cultures) days post-treatment. The optimized concentration/dose of Nfx and XRT were used for combination experiments. MB cells were treated with vehicle, Nfx (20 μg/ml), XRT (5 or 10 Gy) or both and the effect on cell viability, apoptosis, ROS levels and catalase activity was measured. Cell viability was measured with CellTiter Glo kit (Promega). Apoptosis was analyzed by flow cytometry (Annexin-V staining) and caspase 3/7 activity was measured using Caspase-Glo Assay (Promega). The expression of c-PARP was determined by Western blot analysis. Results revealed that Nfx and XRT combination significantly increased MB cell growth inhibition when compared to the effect of either Nfx or XRT. The anti-proliferative effect of Nfx and XRT combination was accompanied by the activation of apoptosis as determined by increased annexin V staining and caspase 3/7 activity. An increase in levels of ROS was also observed following combination treatment with Nfx and XRT. Notably, catalase activity was unaltered following the treatment with single or double agents suggesting a contribution of impairment in the redox system in causing MB cell growth inhibition. This pre-clinical study demonstrates that Nfx potentially enhances the therapeutic efficacy of radiation in MB cell lines and primary cultures. To further delineate the underlying mechanisms and to identify other potential candidates modulated by this combination therapy we are undertaking a molecular profiling approach using Affimetrix gene expression arrays. Citation Format: Don Eslin, Chris M. Lee, Giselle Sholler, Amy Smith, Robert M. Sutphin, Dennis A. Steindler, Ping Zhao, Shanshan Wang, Umesh T. Sankpal, Sarah Connelly, Riyaz Basha. Nifurtimox and radiation combination increases reactive oxygen species and induces apoptosis in medulloblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B11.

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