Robert M. Sutphin

Supplementary Data from Blockade of MGMT Expression by O6 Benzyl Guanine Leads to Inhibition of Pancreatic Cancer Growth and Induction of Apoptosis

Pulmonary vein stenosis results from a proliferative process that leads to the progressive obstruction of venous return to the left atrium. It is often resistant to catheterization and surgical based interventions and is frequently fatal when encountered in its severe form. Here, we describe three patients with severe, primary pulmonary vein stenosis that was progressing despite aggressive conventional management strategies. All three patients were initiated on combination chemotherapy with imatinib and sirolimus, drugs which have been previously shown to independently have potential benefit against PVS. Soon after the initiation of these therapies, all three patients experienced a stabilization of their disease process and clinical improvement. All three patients remain alive, with tolerable side effects from the medications. Although early in our experience and with only a small number of patients, combination chemotherapy with imatinib and sirolimus shows promise and merits further investigation as a therapeutic option for this aggressive disease.

Chemotherapeutic regimens used for the treatment of Neuroblastoma (NB) cause long‐term side effects in pediatric patients. NB arises in immature sympathetic nerve cells and primarily affects infants and children. A high rate of relapse in high‐risk neuroblastoma (HRNB) necessitates the development of alternative strategies for effective treatment. This study investigated the efficacy of a small molecule, tolfenamic acid (TA), for enhancing the anti‐proliferative effect of 13 cis‐retinoic acid (RA) in HRNB cell lines. LA1‐55n and SH‐SY5Y cells were treated with TA (30 μM) or RA (20 μM) or both (optimized doses, derived from dose curves) for 48 h and tested the effect on cell viability, apoptosis and selected molecular markers (Sp1, survivin, AKT and ERK1/2). Cell viability and caspase activity were measured using the CellTiter‐Glo and Caspase‐Glo kits. The apoptotic cell population was determined by flow cytometry with Annexin‐V staining. The expression of Sp1, survivin, AKT, ERK1/2 and c‐PARP was evaluated by Western blots. The combination therapy of TA and RA resulted in significant inhibition of cell viability (p < 0.0001) when compared to individual agents. The anti‐proliferative effect is accompanied by a decrease in Sp1 and survivin expression and an increase in apoptotic markers, Annexin‐V positive cells, caspase 3/7 activity and c‐PARP levels. Notably, TA + RA combination also caused down regulation of AKT and ERK1/2 suggesting a distinct impact on survival and proliferation pathways via signaling cascades. This study demonstrates that the TA mediated inhibition of Sp1 in combination with RA provides a novel therapeutic strategy for the effective treatment of HRNB in children.

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.

Purpose 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. Methods Patients received mifamurtide 2 mg/m² intravenously twice‐weekly ×12 weeks, then weekly ×24 weeks with and without chemotherapy. Serum concentration‐time profiles were collected. Adverse events within 24 hours of drug administration were classified as infusion‐related adverse events (IRAE); other AEs and overall survival (OS) were assessed. Results The study began therapy in January 2008; the last patient completed therapy in October 2012. Two hundred five patients were enrolled; median age was 16.0 years and 146/205 (71%) had active disease. Mifamurtide serum concentrations declined rapidly in the first 30 minutes post‐infusion, then in a log‐linear manner 2–6 hours post‐dose; t_1/2 was 2 hours. There were no readily apparent relationships between age and BSA‐normalized clearance, half‐life, or pharmacodynamic effects, supporting the dose of 2 mg/m² mifamurtide across the age range. Patients reported 3,679 IRAE after 7,482 mifamurtide infusions. These were very rarely grade 3 or 4 and most commonly included chills + fever or headache + fatigue symptom clusters. One‐ and 2‐year OS was 71.7% and 45.9%. Patients with initial metastatic disease or progression approximated by within 9 months of diagnosis (N = 40) had similar 2‐year OS (39.9%) as the entire cohort (45.9%) Conclusions Mifamurtide had a manageable safety profile; PK/PD of mifamurtide in this patient access study was consistent with prior studies. Two‐year OS was 45.9%. A randomized clinical trial would be required to definitively determine impact on patient outcomes. Pediatr Blood Cancer 2014;61:238–244. © 2013 The Authors. Pediatric Blood & Cancer, published by Wiley Periodicals, Inc.

Medulloblastoma (MB) is the most common malignancy in children arising in the brain. Morbidities associated with intensive therapy are serious concerns in treating MB. Our aim was to identify novel targets and agents with less toxicity for treating MB. Specificity protein 1 (Sp1) transcription factor regulates several genes involved in cell proliferation and cell survival including survivin, an inhibitor of apoptosis protein. We previously showed that tolfenamic acid (TA), a nonsteroidal anti-inflammatory drug, inhibits neuroblastoma cell growth by targeting Sp1. We investigated the anticancer activity of TA using human MB cell lines and a mouse xenograft model. DAOY and D283 cells were treated with vehicle (dimethyl sulfoxide) or TA (5–50 μg/ml), and cell viability was measured at 1–3 days posttreatment. TA inhibited MB cell growth in a time- and dose-dependent manner. MB cells were treated with vehicle or TA (10 μg/ml), and the effect on cell apoptosis was measured. Apoptosis was analyzed by flow cytometry (annexin V staining), and caspase 3/7 activity was determined using Caspase-Glo kit. The expression of Sp1, cleaved poly(ADP-ribose) polymerase (c-PARP), and survivin was determined by Western blot analysis. TA inhibited the expression of Sp1 and survivin and upregulated c-PARP. Athymic nude mice were subcutaneously injected with D283 cells and treated with TA (50 mg/kg, three times per week) for 4 weeks. TA caused a decrease of ~40 % in tumor weight and volume. The tumor growth inhibition was accompanied by a decrease in Sp1 and survivin expression in tumor tissue. These preclinical data demonstrate that TA acts as an anticancer agent in MB potentially targeting Sp1 and survivin.

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 has not been evaluated. TA targets specificity protein (Sp) transcription factors that mediate the expression of several genes associated with cancer including survivin, a key member of inhibitor of apoptosis protein family. Our aim was to test the anti-leukemic efficacy of TA in pre-clinical experiments. The anti-leukemic response of TA was determined using Jurkat and Nalm-6 cell lines. Cells were treated with increasing (25/50/75 μM) concentrations of TA, and cell viability was measured at 24, 48, and 72 h post-treatment. TA showed a steady and consistent decrease in cell viability following a clear dose and time dependent response. Apoptosis and cell cycle analysis was performed using flow cytometry. Results showed a significant increase in the apoptotic fraction (annexin V positive) following TA treatment, while cell cycle phase distribution analysis showed G₀/G₁ arrest. TA-induced apoptosis was further confirmed by examining the activation of caspase 3/7 and the expression of cleaved PARP. TA modulated the expression of critical candidates associated with the early phases of cell cycle and validated its efficacy in causing G₀/G₁ arrest. The Western blot results revealed that TA significantly decreases Sp1 and survivin expression. These results demonstrate that the anti-leukemic response of TA occurs potentially through targeting Sp1 and inhibiting survivin and suggest the efficacy of TA as a novel therapeutic agent for leukemia.