Sarah F. Connelly

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.

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.

Small molecule tolfenamic acid and dietary spice curcumin treatment enhances antiproliferative effect in pancreatic cancer cells via suppressing Sp1, disrupting NF-kB translocation to nucleus and cell cycle phase distribution.

Combination of dietary/herbal spice curcumin (Cur) and COX inhibitors has been tested for improving therapeutic efficacy in pancreatic cancer (PC). The objective of this study was to identify agent with low toxicity and COX-independent mechanism to induce PC cell growth inhibition when used along with Cur. Anticancer NSAID, tolfenamic acid (TA) and Cur combination were evaluated using PC cell lines. L3.6pl and MIA PaCa-2 cells were treated with Cur (5-25μM) or TA (25-100μM) or combination of Cur (7.5μM) and TA (50μM). Cell viability was measured at 24-72h posttreatment using CellTiter-Glo kit. While both agents showed a steady/consistent effect, Cur+TA caused higher growth inhibition. Antiproliferative effect was compared with COX inhibitors, Ibuprofen and Celebrex. Cardiotoxicity was assessed using cordiomyocytes (H9C2). The expression of Sp proteins, survivin and apoptotic markers (western blot), caspase 3/7 (caspase-Glo kit), Annexin-V staining (flow cytometry), reactive oxygen species (ROS) and cell cycle phase distribution (flow cytometry) was measured. Cells were treated with TNF-α, and NF-kB translocation from cytoplasm to nucleus was evaluated (immunofluorescence). When compared to individual agents, combination of Cur+TA caused significant increase in apoptotic markers, ROS levels and inhibited NF-kB translocation to nucleus. TA caused cell cycle arrest in G0/G1, and the combination treatment showed mostly DNA synthesis phase arrest. These results suggest that combination of Cur+TA is less toxic and effectively enhance the therapeutic efficacy in PC cells via COX-independent mechanisms.

Targeting the yin and the yang: combined inhibition of the tyrosine kinase c-Src and the tyrosine phosphatase SHP-2 disrupts pancreatic cancer signaling and biology in vitro and tumor formation in vivo.

Objectives Although c-Src (Src) has emerged as a potential pancreatic cancer target in preclinical studies, Src inhibitors have not demonstrated a significant therapeutic benefit in clinical trials. The objective of these studies was to examine the effects of combining Src inhibition with inhibition of the protein tyrosine phosphatase SHP-2 in pancreatic cancer cells in vitro and in vivo. Methods SHP-2 and Src functions were inhibited by siRNA or small molecule inhibitors. The effects of dual Src/SHP-2 functional inhibition were evaluated by Western blot analysis of downstream signaling pathways; cell biology assays to examine caspase activity, viability, adhesion, migration, and invasion in vitro; and an orthotopic nude mouse model to observe pancreatic tumor formation in vivo. Results Dual targeting of Src and SHP-2 induces an additive or supra-additive loss of phosphorylation of Akt and ERK-1/2 and corresponding increases in expression of apoptotic markers, relative to targeting either protein individually. Combinatorial inhibition of Src and SHP-2 significantly reduces viability, adhesion, migration, and invasion of pancreatic cancer cells in vitro and tumor formation in vivo, relative to individual Src/SHP-2 inhibition. Conclusions These data suggest that the antitumor effects of Src inhibition in pancreatic cancer may be enhanced through simultaneous inhibition of SHP-2.

Tolfenamic acid suppresses cytochrome P450 2E1 expression in mouse liver

Non-steroidal anti-inflammatory drugs (NSAIDs) play a significant role in the chemoprevention of cancer. We recently showed the chemopreventive response of a NSAID, 2-[(3-chloro-2-methylphenyl)amino]benzoic acid) known as tolfenamic acid (TA) in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors in rats. Pre-clinical studies showed that TA inhibits Specificity protein (Sp) transcription factors and acts as an anti-cancer agent in several cancer models; however the pertinent mechanisms associated with its chemopreventive response in esophageal cancer are not known. Since the bioactivation of carcinogens through cytochrome P450 (CYP) is critical for the induction of cancer, we have studied the effect of TA on critical CYP isozymes in mouse liver samples. Athymic nude mice were treated with vehicle (corn oil) or TA (50 mg kg(-1), 3 times per week) for 4 weeks. Protein extracts (whole cell lysates and microsomal fractions) were prepared from liver tissue and the expression of various CYP isozymes was determined by Western blot analysis. Rat (Sprague-Dawley) livers were harvested and primary hepatocyte cultures were treated with vehicle (DMSO) or TA (50 μM) and cell viability was assessed at 2 and 5 days post-treatment. TA caused remarkable decrease in the expression of CYP2E1 in both liver lysates and sub-cellular fraction, while its response on other tested isozymes was marginal. TA did not affect the body weight of animals (mice) and viability of rat hepatocytes. These results demonstrate that TA modulates the expression of CYP2E1 which is associated with the bioactivation of carcinogens without causing apparent toxicity. These data suggest that TA-induced inhibition of CYP2E1 attenuates the bioactivation of carcinogens potentially leading to the chemoprevention of NMBA-induced esophageal tumorigenesis in rats.

Abstract 4818: Tolfenamic acid and curcumin treatment induces pancreatic cancer cell growth inhibition via suppressing Sp1 expression, NF-kB translocation to nucleus

Pancreatic cancer (PC) is an aggressive malignancy. The current treatment options have limited response in addressing poor prognosis and low survival rate. Hence it is important to identify novel agents and strategies for effective treatment. Previously the combination of phytochemical, curcumin (Cur) and cyclooxygenase (COX) inhibitor celecoxib was tested for improving therapeutic efficacy in PC models. The objective of current study is to identify a combination treatment involving a low toxic small molecule and a phytochemical with anti-cancer properties to inhibit PC cell growth. Experiments were also conducted to understand potential mechanisms associated with this combination. We tested the combination of an anti-cancer non-steroidal anti-inflammatory drug (NSAID), Tolfenamic Acid (TA) and Cur using PC cell lines, L3.6 and MIA PaCa-2. Cells were treated with 5-25 μM of Cur or 25-100 μM of TA or combination of Cur (7.5 μM) and TA (50 μM). Effect on cell viability was measured at 24, 48 and 72 h post-treatment using CellTiter-Glo kit. While the two agents showed anti-proliferative effect, Cur and TA combination caused higher growth inhibition. The cell growth inhibition was compared with two COX inhibitors, ibuprofen and celecoxib and the cardiotoxicity was assessed using cordiomyocytes (H9C2). TA showed significantly less cytotoxicity in cardiomyocytes when compared to celecoxib. The expression of transcription factors, Specificity protein1 (Sp1) and NF-kB, and an inhibitor of apoptosis family protein, survivin, were determined by Western blot analysis. The expression of NF-kB, Sp1 and survivin was decreased by combination treatment. The levels of reactive oxygen species (ROS) were also measured in flowcytometer. To evaluate the effect of these agents on apoptosis, the activity of caspase 3/7 was measured with caspase-Glo kit; apoptotic cell population was evaluated by Annexin-V staining (flow cytometry); and c-PARP expression was determined by Western blot analysis. When compared to individual agents, the combination treatment caused a significant increase in ROS levels and apoptotic markers. L3.6 and MIA PaCa-2 cells were treated with TNF-a to induce NF-kB translocation from cytoplasm to nucleus and the effect of individual (TA or Cur) and combined treatment (TA+Cur) on NF-kB translocation from cytoplasm to nucleus was evaluated by immunofluorescence. When compared to individual agents, the combination treatment caused a significant decrease in NF-kB translocation to nucleus. Cell cycle phase distribution was measured using flow cytometry. The combination treatment showed mostly DNA synthesis phase arrest; however TA caused cell cycle arrest in early phase (G0/G1). These results demonstrate that combination of Cur and TA effectively inhibits PC cell growth via inducing apoptosis and modulating cell cycle phase distribution. Citation Format: Riyaz M. Basha, Sarah F. Connelly, Ganji Purnachandra, Umesh T. Sankpal, Hassaan Patel, Jamboor K. Vishwanatha, Sagar Shelake, Leslie Tabor-Simecka1, Mamoru Shoji, Jerry Simecka W. Simecka, Bassel El-Rayes. Tolfenamic acid and curcumin treatment induces pancreatic cancer cell growth inhibition via suppressing Sp1 expression, NF-kB translocation to nucleus. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4818.

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 4216: Combination of anti-cancer small molecule tolfenamic acid and curcumin or curcumin analog EF31 effectively inhibits pancreatic cancer cell growth

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug is known to inhibit human cancer cells and mouse tumor growth in some cancer models. TA is known to target the transcription factor, specificity protein1 (Sp1) that mediates the expression of several genes associated with cancer including survivin, a key member of inhibitor of apoptosis proteins (IAP) family. It is currently in phase I clinical trials for treating upper gastro-intestinal cancer patients along with radiation. Curcumin (Cur) is an aromatic constituent of the plant curcuma longa (turmeric) which is extensively studied in some malignancies including breast, pancreatic and colon cancers. Even though, Cur shows a broad spectrum of anti-cancer activity in pre-clinical studies, its clinical application is greatly affected by its low bioavailability. Hence the strategies to improve the response to Cur soared by synthesizing and testing analogs. Recently our group showed that EF31 impacts the DNA methylation and causes anti-proliferative effect in pancreatic cancer cell liens and inhibits tumor growth in mice xenografts. We have evaluated the therapeutic efficacy of Cur and a Cur analog EF31 in combination with an anti-cancer small molecule, TA using human pancreatic cancer cell lines, MiaPaCa2 and L3.6 pl. MiaPaCa2 and L3.6 pl cells were treated with increasing concentrations of TA (25-100 µM) or Cur (5-25 µM) or EF31 (0.2-5 µM) or combination of optimized concentrations of TA (50 µM) and Cur (7.5 µM) or EF31 (0.5 µM) and the cell viability was measured at 24, 48, and 72 h post-treatment. All agents showed a steady and consistent decrease in cell viability following a clear dose and time-dependent response while the combination of TA and Cur or EF31 showed higher growth inhibition. Apoptosis and cell cycle analysis was performed using flow cytometry. Results showed a significant increase in the apoptotic fraction (annexin V positive) following combination treatment when compared to individual effect. TA caused cell cycle arrest in G/G1 and the combination treatment showed both G/G1 and G2 arrest. The activation of apoptosis was further confirmed by examining the activation of caspases (caspase 3/7, 8 and 9) and the expression of cleaved PARP. The Western blot results revealed that TA significantly decreased Sp1 and survivin expression and the combination of TA and Cur or TA and EF31 significantly modulated the expression of critical candidates associated with cell cycle. When compared to Cur, its synthetic analog EF31 showed higher efficacy in both individual and combination studies. These pre-clinical results demonstrate that the combination of anti-cancer NSAID, tolfenamic acid and curcumin or curcumin analogs may enhance the therapeutic efficacy in pancreatic cancer. Studies to better understand the underlying mechanisms by performing molecular profiling are currently under investigation. Citation Format: Riyaz Basha, Sarah F. Connelly, Ganji Purnachandra Nagaraju, Umesh T. Sankpal, Mamoru Shoji, Omar Kayaleh, Bassel El-Rayes. Combination of anti-cancer small molecule tolfenamic acid and curcumin or curcumin analog EF31 effectively inhibits pancreatic cancer cell growth. [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 4216. doi:10.1158/1538-7445.AM2014-4216

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 2859: Dual targeting of protein tyrosine kinase c-Src and protein tyrosine phosphatase SHP-2 is a novel therapeutic strategy that induces potent inhibition of pancreatic cancer cell viability in vitro and tumor progression in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pancreatic adenocarcinoma is a deadly human malignancy, with over 95% of patients succumbing within five years. The majority of pancreatic cancer deaths are caused by metastatic dissemination of the primary tumor. The only curative treatment is surgical resection, which is limited by tumor stage, and the standard chemotherapeutic option, gemcitabine, offers only modest survival and quality of life benefits. There is a need for a greater understanding of pancreatic cancer biology and development of novel therapies to inhibit tumor progression and metastasis. The non-receptor protein tyrosine kinase, c-Src (Src) has emerged as a potential target for the treatment of pancreatic cancer, having been demonstrated to promote a number of tumorigenic processes. Src is overexpressed and/or aberrantly activated in greater than 70% of human pancreatic cancers. In addition, SHP-2, a non-receptor protein tyrosine phosphatase, regulates Src family kinase activity. Evidence has established clinical relevance for SHP-2 in human diseases. A recent study demonstrated that inhibition of SHP-2 abrogates in vitro and in vivo angiogenesis and inhibits activity of the MAPK/Erk1/2 and PI3K/Akt survival pathways. SHP-2 and Src function as key signaling intermediates downstream of growth factor receptors, cytokine receptors, and integrins, and are crucial for activation of downstream cascades of tumor progression and metastasis. The purpose of this study was to determine the importance of SHP-2 and Src, for pancreatic cancer signaling, cell biology, tumorigenicity, and metastasis. We have demonstrated elevated SHP-2 expression and phosphorylation in pancreatic cancer cells, relative to normal pancreatic cells. Phosphorylation of SHP-2 in pancreatic cancer cells occurs in a Src kinase-dependent fashion. Functional inhibition of SHP-2 and Src, individually or in combination, resulted in reduced ERK-1/2 phosphorylation, increased cleavage of caspase 3 and PARP, increased expression of the pro-apoptotic protein Bax, and decreased levels of the anti-apoptotic protein Bcl-xL. These results were mirrored by decreased viability and increased caspase activity in the presence of Src and SHP-2 inhibition. Finally, treatment of mice with small molecule inhibitors of Src or SHP-2, produced markedly smaller pancreatic tumors relative to controls in an orthotopic nude mouse model and dual treatment exacerbated this effect. The potential use for the Src and SHP-2 signaling axes as therapeutic targets remains largely untapped. These data suggest the importance of SHP-2 in pancreatic cancer cell biology and its potential value as a therapeutic target. Our studies suggest that the anti-tumor/anti-metastatic effects of Src inhibition may be improved through simultaneous inhibition of SHP-2. 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 2859. doi:1538-7445.AM2012-2859