Activating Notch with a Notch agonist peptide induces apoptosis in AML patient samples.

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. 205 patients were enrolled; median age was 16.5 years and 143/204 (72%) had active disease. Mifamurtide serum concentrations declined rapidly in the first 30 minutes post-infusion, then in a loglinear manner 2–6 hours post-dose; t1/2 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 3,415 IRAE after 7,122 mifamurtide infusions. These were very rarely grade 3 or 4 and most commonly included chills+fever or headache+fatigue symptom clusters. One and two year OS was 70.6% and 41.4%. Patients with initial metastatic disease or progression approximated by within 9 months of diagnosis (N=40) had similar 2-year OS (38.8%) as the entire cohort (41.4%) 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 41.4%. A randomized clinical trial would be required to definitively determine impact on patient outcomes.

These data demonstrate that early lymphocyte recovery represents a significant prognostic indicator for osteosarcoma. Early identification and risk stratification therapy based on the ALC-14 threshold may improve outcomes and our knowledge of this disease.

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.

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.

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: We sought to determine whether administration of a MGMT blocker, O 6 -benzyl guanine (O 6 BG), at an optimal biological dose alone or in combination with gemcitabine inhibits human pancreatic cancer cell growth. Experimental Design: Human pancreatic cancer L3.6pl and PANC1 cells were treated with O 6 BG, either alone or in combination with gemcitabine, and the therapeutic efficacy and biological activity of these drug combinations were investigated. Results: O 6 BG sensitized pancreatic cancer cells to gemcitabine. Protein and mRNA expression of MGMT, cyclin B1, cyclin B2, cyclin A, and ki-67 were significantly decreased in the presence of O 6 BG. In sharp contrast, protein expression and mRNA message of p21 cip1 were significantly increased. Interestingly, O 6 BG increases p53-mediated p21 cip1 transcriptional activity and suppresses cyclin B1. In addition, our results indicate that p53 is recruited to p21 promoter. Furthermore, an increase in p21 cip1 and a decrease in cyclin transcription are p53 dependent. The volume of pancreatic tumors was reduced by 27% in mice treated with gemcitabine alone, by 47% in those treated with O 6 BG alone, and by 65% in those mice given combination. Immunohistochemical analysis showed that O 6 BG inhibited expression of MGMT and cyclins, and increased expression of p21 cip1 . Furthermore, there was a significant decrease in tumor cell proliferation and an increase in tumor cell apoptosis. Conclusions: Collectively, our results show that decreased MGMT expression is correlated with p53 activation, and significantly reduced primary pancreatic tumor growth. These findings suggest that O 6 BG either alone or in combination with gemcitabine may provide a novel and effective approach for the treatment of human pancreatic cancer. (Clin Cancer Res 2009;15(19):6087-95)

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.