Targeted Molecular Therapeutic Options for Hepatocellular Carcinoma.

Liver cancer is the 6th leading cause of cancer related deaths in the US even though it ranks 14th in incidence. More men are diagnosed with liver cancer than women, and the number of projected deaths among men (20,020) is almost double that among women (10,140) in the US. Infections like hepatitis and metabolic conditions like obesity are believed to be major risk factors for the onset of liver cancer. Hepatocellular carcinoma (HCC), the most common type of liver cancer, accounts for 75% of all cases. Chemotherapy has not been effective in treating HCC. Targeted therapies are being used in advanced HCC patients due to a better survival and less side effects when compared to traditional chemotherapy. Therapeutic agents targeting the regulators of growth factor signaling pathways and the mediators of downstream signaling-for example, inhibitors of the tyrosine kinase receptor-are used as targeted molecular therapies. Kinase inhibitors that modulate growth signals, such as sorafenib and lenvatinib, are commonly employed in targeted molecular therapy for HCC patients. This review covers these agents, highlighting modes of action and providing details on clinical trials.

Next generation sequencing and functional pathway analysis to understand the mechanism of action of copper-tolfenamic acid against pancreatic cancer cells.

Anti-cancer activity of tolfenamic acid (TA) in preclinical models for pancreatic cancer (PaCa) is well established. Since the dosage for anti-cancer actions of TA is rather high, we recently demonstrated that IC50 values of Copper-TA are 30-80% less than TA in 12 cancer cell lines. This study elucidates the underlying mechanisms of Copper-TA in PaCa cells. Control and Copper-TA (IC50) treated PaCa cells were processed by next-generation sequencing (NGS) to determine differentially expressed genes using HTG EdgeSeq Oncology Biomarker panel. Ingenuity Pathway Analysis (IPA®) was used to identify functional significance of altered genes. The conformational studies for assessing the expression of key regulators and genes were conducted by Western blot and qPCR. IPA® identified several networks, regulators, as well as molecular and cellular functions associated with cancer. The top 5 molecular and cellular functions affected by Cu-TA treatment were cell death and survival, cellular development, cell growth and proliferation, cell cycle and cellular movement. The expression of top upstream regulators was confirmed by Western blot analysis while qPCR results of selected genes demonstrated that Copper-TA is efficacious at lower doses than TA. Results suggest that Copper-TA alters genes/key regulators associated with cancer and potentially serve as an effective anti-cancer agent.

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.

Clotam enhances anti-proliferative effect of vincristine in Ewing sarcoma cells.

Current therapeutic strategies used in Ewing sarcoma (ES) especially for relapsed patients have resulted in modest improvements in survival over the past 20 years. Combination therapeutic approach presents as an alternative to overcoming drug resistance in metastatic ES. This study evaluated the effect of Clotam (tolfenamic acid or TA), a small molecule and inhibitor of Specificity protein1 (Sp1) and survivin for sensitizing ES cell lines to chemotherapeutic agent, vincristine (VCR). ES cells (CHLA-9 and TC-32) were treated with TA or VCR or TA + VCR (combination), and cell viability was assessed after 24/48/72 h. Effect of TA or VCR or TA + VCR treatment on cell cycle arrest and apoptosis were evaluated using propidium iodide, cell cycle assay and Annexin V flow cytometry respectively. The apoptosis markers, caspase 3/7 (activity levels) and cleaved-PARP (protein expression) were measured. Cardiomyocytes, H9C2 were used as non-malignant cells. While, all treatments caused time- and dose-dependent inhibition of cell viability, interestingly, combination treatment caused significantly higher response (~ 80% inhibition, p < 0.05). Cell viability inhibition was accompanied by inhibition of Sp1 and Survivin. TA + VCR treatment significantly (p < 0.05) increased caspase 3/7 activity which strongly correlated with upregulated c-PARP level and Annexin V staining. Cell cycle arrest was observed at G0/G1 (TA) or G2/M (VCR and TA + VCR). All treatments did not cause cytotoxicity in H9C2 cells. These results suggest that TA could enhance the anti-cancer activity of VCR in ES cells. Therefore, TA + VCR combination could be further tested to develop as safe/effective therapeutic strategy for treating ES.

Copper-tolfenamic acid: evaluation of stability and anti-cancer activity.

The non-steroidal anti-inflammatory drug, Tolfenamic acid (TA) acts as an anti-cancer agent in several adult and pediatric cancer models. Copper (Cu) is an important element with multiple biological functions and has gained interest in medical applications. Recently, [Cu(TA)2(bpy)] (Cu-TA) has been synthesized in order to enhance therapeutic activity. In this study, we synthesized Cu-TA using an established method, characterized it by UV visible spectroscopy and Fourier-transform infrared spectroscopy (FTIR), and tested its anti-cancer activity using twelve cell lines representing various cancers, such as Ewing sarcoma, glioblastoma, medulloblastoma, neuroblastoma, pancreatic and prostate. The anti-proliferative activity of Cu-TA was determined at 48 h post-treatment and compared with the parental compound, TA. The IC50 values were calculated using GraphPad Prism software. The biological stability of Cu-TA was evaluated using twelve-month-old powder and six-month-old stock solution. Cardiomyocytes (H9C2) were used to test the cytotoxicity in non-malignant cells. Cu-TA showed higher anti-proliferative activity, and the IC50 values were 30 to 80% lower when compared with TA. H9C2 cells were non-responsive to Cu-TA, suggesting that it is selective towards malignant cells. Comparison of the twelve-month-old powder and six-month-old stock solution using the Panc1 cell line showed similar IC50 values (<5% variation), confirming the stability of Cu-TA either in powder or solution form. These findings demonstrate the potential of Cu-TA as an effective anti-cancer agent. Further studies to delineate the detailed mechanism of action of Cu-TA for specific cancer model are underway.

Impact of the Microbiome on the Immune System.

Higher organisms are all born with general immunity as well as with, increasingly, more specific immune systems. All immune mechanisms function with the intent of aiding the body in defense against infection. Internal and external factors alike have varying effects on the immune system, and the immune response is tailored specifically to each one. Accompanying the components of the human innate and adaptive immune systems are the other intermingling systems of the human body. Increasing understanding of the body's immune interactions with other systems has opened new avenues of study, including that of the microbiome. The microbiome has become a highly active area of research over the last 10 to 20 years since the NIH began funding the Human Microbiome Project (HMP), which was established in 2007. Several publications have focused on the characterization, functions, and complex interplay of the microbiome as it relates to the rest of the body. A dysfunction between the microbiome and the host has been linked to various diseases including cancers, metabolic deficiencies, autoimmune disorders, and infectious diseases. Further understanding of the microbiome and its interaction with the host in relation to diseases is needed in order to understand the implications of microbiome dysfunction and the possible use of microbiota in the prevention of disease. In this review, we have summarized information on the immune system, the microbiome, the microbiome's interplay with other systems, and the association of the immune system and the microbiome in diseases such as diabetes and colorectal cancer.

Novel Survivin Inhibitor for Suppressing Pancreatic Cancer Cells Growth via Downregulating Sp1 and Sp3 Transcription Factors.

BACKGROUND/AIMS: Targeting survivin, an anti-apoptotic protein and mitotic regulator, is considered as an effective therapeutic option for pancreatic cancer (PaCa). Tolfenamic acid (TA) showed anti-cancer activity in pre-clinical studies. A recent discovery demonstrated a copper(II) complex of TA (Cu-TA) can result in higher activity. In this study, the ability of Cu-TA to inhibit survivin and its transcription factors, Specificity protein (Sp) 1 and 3 in PaCa cell lines and tumor growth in mouse xenograft model were evaluated. METHODS: Cell growth inhibition was measured in MIA PaCa-2 and Panc1 cells for 2 days using CellTiter-Glo kit. Sp1, Sp3 and survivin expression (by Western blot and qPCR), apoptotic cells and cell cycle phase distribution (by flow cytometry) were evaluated. A pilot study was performed using athymic nude mice [treated with vehicle/Cu-TA (25 or 50 mg/kg) 3 times/week for 4 weeks. RESULTS: The IC50 value for Cu-TA was about half than TA.Both agents repressed the protein expression of Sp1/Sp3/survivin, Cu-TA was more effective than TA. Especially effect on survivin inhibition was 5.2 (MIA PaCa-2) or 6.4 (Panc1) fold higher and mRNA expression of only survivin was decreased. Apoptotic cells increased with Cu-TA treatment in both cell lines, while Panc1 showed both effect on apoptosis and cell cycle (G2/M) arrest. Cu-TA decreased the tumor growth in mouse xenografts (25 mg/kg: 48%; 50 mg/kg: 68%). Additionally, there was no change observed in mice body weights, indicating no overt toxicity was occurring. CONCLUSION: These results show that Cu-TA can serve as an effective survivin inhibitor for inhibiting PaCa cell growth.

Investigational agents to enhance the efficacy of chemotherapy or radiation in pancreatic cancer.

Pancreatic cancer (PC) continues to be a fatal malignancy. With standard treatments having modest impact, alternative courses of actions are being investigated such as enhancing the efficacy of standard treatment through sensitization of PC cells to chemotherapy or radiation. This review emphasizes investigational agents that increase the responses to chemotherapy or radiation in PC models. Our group has extensively investigated on Curcumin (Cur), analogs (EF31, UBS109, and L49H37), nanoparticles and a small molecule Tolfenamic acid (TA) for enhancing therapeutic efficacy in both in vitro and in vivo assays. Cur has a low level of toxicity and promising anti-cancer activity, however, its clinical development has been limited by low bioavailability. Cur analogs and nanoparticles were synthesized to improve Cur's efficacy and bioavailability. These compounds were found to be effective in enhancing the therapeutic effects of chemotherapy in pre-clinical models. Small molecules such as NSAIDs have also been tested for the anti-cancer activity and induction of response of chemotherapy and radiation. Interest in TA, a NSAID, has recently increased due to promising preclinical data demonstrating its anti-cancer properties with minimum toxicity. TA also synergistically increased the response of XRT in PC cells and in an orthotropic mouse model. With strong preclinical evidence, research aimed at developing less toxic therapies for PC using Cur analogues or TA is ready for translation into clinical testing.

Tolfenamic acid-induced alterations in genes and pathways in pancreatic cancer cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) are being tested extensively for their role in the treatment and prevention of several cancers. Typically NSAIDs exhibit anti-tumor activities via modulation of cyclooxygenase (COX)-dependent mechanisms, however, an anti-cancer NSAID tolfenamic acid (TA) is believed to work through COX-independent pathways. Results from our laboratory and others have demonstrated the anti-cancer activity of TA in various cancer models including pancreatic cancer. TA has been shown to modulate certain cellular processes including, apoptosis, reactive oxygen species and signaling. In this study, molecular profiling was performed to precisely understand the mode of action of TA. Three pancreatic cancer cell lines, L3.6pl, MIA PaCa-2, and Panc1 were treated with TA (50 µM for 48 h) and the changes in gene expression was evaluated using the Affymetrix GeneChip Human Gene ST Array platform. Microarray results were further validated using quantitative PCR for seven genes altered by TA treatment in all three cell lines. Functional analysis of differentially expressed genes (2 fold increase or decrease, p < 0.05) using Ingenuity Pathway Analysis software, revealed that TA treatment predominantly affected the genes involved in cell cycle, cell growth and proliferation, and cell death and survival. Promoter analysis of the differentially expressed genes revealed that they are enriched for Sp1 binding sites, suggesting that Sp1 could be a major contributor in mediating the effect of TA. The gene expression studies identified new targets involved in TA's mode of action, while supporting the hypothesis about the association of Sp1 in TA mediated effects in pancreatic cancer.

Targeting specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting Ewing sarcoma cell growth.

Transcription factor Specificity protein 1 (Sp1) and its downstream target survivin (inhibitor of apoptosis protein), play major roles in the pathogenesis of various cancers. Ewing Sarcoma (ES) is a common soft tissue/bone tumor in adolescent and young adults. Overexpression of survivin is also linked to the aggressiveness and poor prognosis of ES. Small molecule Tolfenamic acid (TA) inhibits Sp1 and survivin in cancer cells. In this investigation, we demonstrate a strategy to target Sp1 and survivin using TA and positive control Mithramycin A (Mit) to inhibit ES cell growth. Knock down of Sp1 using small interfering RNA (siRNA) resulted in significant (p < 0.05) inhibition of CHLA-9 and TC-32 cell growth as assessed by CellTiter-Glo assay kit. TA or Mit treatment caused dose/time-dependent inhibition of cell viability, and this inhibition was correlated with a decrease in Sp1 and survivin protein levels in ES cells. Quantitative PCR results showed that Mit treatment decreased the mRNA expression of both survivin and Sp1, whereas TA diminished only survivin but not Sp1. Proteasome inhibitor restored TA-induced inhibition of Sp1 protein expression suggesting that TA might cause proteasome-dependent degradation. Gel shift assay using ES cell nuclear extract and biotinylated Sp1 consensus oligonucleotides confirmed that both TA and Mit decreased DNA-binding activity of Sp1. These results demonstrate that both Mit and TA reduce expression of Sp1 and survivin, disrupt Sp1 DNA-binding and inhibit ES cell proliferation. This investigation suggests that targeting Sp1 and survivin could be an effective strategy for inhibiting ES cell growth.

Association of Sp1 and survivin in epithelial ovarian cancer: Sp1 inhibitor and cisplatin, a novel combination for inhibiting epithelial ovarian cancer cell proliferation.

The expression of specificity protein 1 (Sp1) and survivin was evaluated in clinical specimens of epithelial ovarian cancer (EOC) patients. When compared to normal tissue, EOC samples showed high expression of Sp1 and survivin using qPCR (Sp1: ∼2-fold; survivin: ∼5-fold) and Western blot (Sp1: >2.6-fold; survivin: >100-fold). The Sp1 inhibitor, and anti-cancer small molecule, tolfenamic acid (TA), was tested to enhance the response of Cisplatin (Cis) in EOC cell lines. Cell viability (CellTiter-Glo), combination index (CalcuSyn software), apoptosis (Annexin-V staining), cell cycle analyses (flow cytometry), and reactive oxygen species (flow cytometry) were determined. Cell migration and invasion was assessed using matrigel coated transwell chambers. Agilent Technologies proteomics analysis identified potential signaling pathways involved. The combination of TA (50 µM) and Cis (5 µM) synergistically increased the growth inhibition in ES2 (∼80 %, p < 0.001) and OVCAR-3 (60 %, p < 0.001) cells. TA or TA + Cis treatment in ES2 cells caused cell cycle arrest in G1 Phase (TA) or S-Phase (TA + Cis) and unregulated reactive oxygen species. Invasion and migration was decreased in ES2 cells. Global proteomic profiling showed modulation of proteins associated with oxidative phosphorylation, apoptosis, electron transport chain, DNA damage, and cell cycle proteins. These results demonstrate an association of Sp1 and survivin in EOC and confirm targeting these candidates with TA potentially sensitizes EOC cells to cisplatin.

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.

Combination of tolfenamic acid and curcumin induces colon cancer cell growth inhibition through modulating specific transcription factors and reactive oxygen species.

Curcumin (Cur) has been extensively studied in several types of malignancies including colorectal cancer (CRC); however its clinical application is greatly affected by low bioavailability. Several strategies to improve the therapeutic response of Cur are being pursued, including its combination with small molecules and drugs. We investigated the therapeutic efficacy of Cur in combination with the small molecule tolfenamic acid (TA) in CRC cell lines. TA has been shown to inhibit the growth of human cancer cells in vitro and in vivo, via targeting the transcription factor specificity protein1 (Sp1) and suppressing survivin expression. CRC cell lines HCT116 and HT29 were treated with TA and/or Cur and cell viability was measured 24-72 hours post-treatment. While both agents caused a steady reduction in cell viability, following a clear dose/ time-dependent response, the combination of TA+Cur showed higher growth inhibition when compared to either single agent. Effects on apoptosis were determined using flow cytometry (JC-1 staining to measure mitochondrial membrane potential), Western blot analysis (c-PARP expression) and caspase 3/7 activity. Reactive oxygen species (ROS) levels were measured by flow cytometry and the translocation of NF-kB into the nucleus was determined using immunofluorescence. Results showed that apoptotic markers and ROS activity were significantly upregulated following combination treatment, when compared to the individual agents. This was accompanied by decreased expression of Sp1, survivin and NF-kB translocation. The combination of TA+Cur was more effective in HCT116 cells than HT29 cells. These results demonstrate that TA may enhance the anti-proliferative efficacy of Cur in CRC cells.

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.

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.

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.

Expression of specificity protein transcription factors in pancreatic cancer and their association in prognosis and therapy.

Pancreatic cancer is an aggressive malignancy with poor prognosis. Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths in the United States. Due to the aggressive nature of this malignancy, there is a serious concern for identifying effective targets, and adopting novel strategies for therapy. Members of the Specificity Protein (Sp) family of transcription factors, Sp1, Sp3, and Sp4 regulate the expression of a number of genes associated with cancer cell proliferation, differentiation, and metastasis. Sp1 levels are upregulated in pancreatic cancer cell lines, and surgically resected human pancreatic adenocarcinoma. Sp1 overexpression in tumor tissues is associated with aggressive disease, poor prognosis and inversely correlated with survival. Sp1 is also known to affect angiogenesis by regulating the expression of vascular endothelial growth factor and its receptors. Results from clinical studies suggest Sp1 as new biomarker to identify aggressive pancreatic ductal adenocarcinoma. The pharmacological inhibition of Sp1 using agents such as celecoxib, mithramycin, curcumin, and tolfenamic acid has showed promising results in pre-clinical studies and demonstrated Sp transcription factors as potential targets for pancreatic cancer therapy. This review summarizes studies showing the association of Sp proteins with this malignancy, with a special emphasis on pre-clinical studies that tested strategies to target Sp transcription factors for inhibiting human pancreatic cancer cell proliferation and tumor growth in laboratory animals. The results showed remarkable efficacy and suggest that such approaches have the potential for high success in developing clinically relevant strategies for treating pancreatic cancer.