Combination Chemotherapy in Severe Pulmonary Vein Stenosis-A Case Series.

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.

Combination of clotam and vincristine enhances anti-proliferative effect in medulloblastoma cells.

Medulloblastoma (MB) is characterized by highly invasive embryonal neuro-epithelial tumors that metastasize via cerebrospinal fluid. MB is difficult to treat and the chemotherapy is associated with significant toxicities and potential long-term disabilities. Previously, we showed that small molecule, clotam (tolfenamic acid: TA) inhibited MB cell proliferation and tumor growth in mice by targeting, survivin. Overexpression of survivin is associated with aggressiveness and poor prognosis in several cancers, including MB. The aim of this study was to test combination treatment involving Vincristine® (VCR), a standard chemotherapeutic drug for MB and TA against MB cells. DAOY and D283 MB cells were treated with 10 µg/mL TA or VCR (DAOY: 2 ng/mL; D283: 1 ng/mL) or combination (TA + VCR). These optimized doses were lower than individual IC50 values. The effect of single or combination treatment on cell viability (CellTiterGlo kit), Combination Index (Chou-Talalay method based on median-drug effect analysis), activation of apoptosis and cell cycle modulation (by flow cytometry using Annexin V and propidium iodide respectively) and the expression of associated markers including survivin (Western immunoblot) were determined. Combination Index showed moderate synergistic cytotoxic effect in both cells. When compared to individual agents, the combination of TA and VCR increased MB cell growth inhibition, induced apoptosis and caused cell cycle (G2/M phase) arrest. Survivin expression was also decreased by the combination treatment. TA is effective for inducing the anti-proliferative response of VCR in MB cells. MB has four distinct genetic/molecular subgroups. Experiments were conducted with MB cells representing two subgroups (DAOY: SHH group; D283: group 4/3). TA-induced inhibition of survivin expression potentially destabilizes mitotic microtubule assembly, sensitizing MB cells and enhancing the efficacy of VCR.

Combination of 13 cis-retinoic acid and tolfenamic acid induces apoptosis and effectively inhibits high-risk neuroblastoma cell proliferation.

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 (30µM) or RA (20µM) or both (optimized doses, derived from dose curves) for 48h 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.

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 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.

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.

Anticancer activity of tolfenamic acid in medulloblastoma: a preclinical study.

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.

Notch activation inhibits AML growth and survival: a potential therapeutic approach.

Although aberrant Notch activation contributes to leukemogenesis in T cells, its role in acute myelogenous leukemia (AML) remains unclear. Here, we report that human AML samples have robust expression of Notch receptors; however, Notch receptor activation and expression of downstream Notch targets are remarkably low, suggesting that Notch is present but not constitutively activated in human AML. The functional role of these Notch receptors in AML is not known. Induced activation through any of the Notch receptors (Notch1-4), or through the Notch target Hairy/Enhancer of Split 1 (HES1), consistently leads to AML growth arrest and caspase-dependent apoptosis, which are associated with B cell lymphoma 2 (BCL2) loss and enhanced p53/p21 expression. These effects were dependent on the HES1 repressor domain and were rescued through reexpression of BCL2. Importantly, activated Notch1, Notch2, and HES1 all led to inhibited AML growth in vivo, and Notch inhibition via dnMAML enhanced proliferation in vivo, thus revealing the physiological inhibition of AML growth in vivo in response to Notch signaling. As a novel therapeutic approach, we used a Notch agonist peptide that led to significant apoptosis in AML patient samples. In conclusion, we report consistent Notch-mediated growth arrest and apoptosis in human AML, and propose the development of Notch agonists as a potential therapeutic approach in AML.

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.

Prognostic significance of early lymphocyte recovery in pediatric osteosarcoma.

BACKGROUND: 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. PROCEDURES: We reviewed data of 19 consecutive patients treated for osteosarcoma at our institution from 1997 to 2007. After initial chemotherapy, patients were separated into two groups: early versus late lymphocyte recovery, using a threshold absolute lymphocyte count of ≥ 800 cells/µl on day 14 (ALC-14). RESULTS: The 5-year overall survival (OS) for our cohort was 73.7% [± 10.1 standard error (SE)]. Thirteen patients (68%) had an ALC-14 ≥ 800 cells/µl, with 12/13 alive and 5-year OS of 92.3% (± 7.4 SE). In contrast, six patients (32%) had an ALC-14 < 800 cells/µL, with 1/6 alive and 5-year OS of 33.3% (± 19.2 SE). The difference is statistically significant (P = 0.0013, log-rank test). Two patients presented with multifocal disease at diagnosis, had late lymphocyte recovery and died. One patient presented with metastatic disease, had early lymphocyte recovery and is alive. Six patients developed relapsed disease with a 5-year OS of 33.3% (± 19.2 SE). The majority (5/6) of patients with relapsed disease died while on active therapy. The only survivor in this group had an ALC-14 > 800 cells/µl and recently completed relapse therapy. CONCLUSIONS: 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.

Blockade of MGMT expression by O6 benzyl guanine leads to inhibition of pancreatic cancer growth and induction of apoptosis.

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.