Tolfenamic acid inhibits neuroblastoma cell proliferation and induces apoptosis: A novel therapeutic agent for neuroblastoma

Abstract

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.

Cellular and Organismal Toxicity of the Anti-Cancer Small Molecule, Tolfenamic Acid: a Pre-Clinical Evaluation

“…2). Cancer cell growth inhibition was accompanied with a decrease in the expression of both Sp1 and survivin in all three pancreatic cancer cell lines, which is also consistent with published pre-clinical work on pancreatic [26] and other cancers [30,32,35,36,37]. Notably, non-malignant hTERT-HPNE cells do not express survivin but do express Sp1, but TA treatment did not affect the expression of Sp1.…”

“…As expected, TA caused minimal effect on differentiated NB cells when compared to un-differentiated SH-SY5Y cells. Recent studies from our group demonstrated that TA induces apoptotic pathways and disrupts cell cycle progression in cancer cells [30,32,35,36,37]. It is plausible that apoptotic and cell cycle processes/pathways are potentially inactive in differentiated cells, thereby reducing the effect of TA in RA treated (differentiated) SH-SY5Y cells.…”

“…Recently we showed that TA inhibits neuroblastoma cells SH-SY5Y and LA 155n by targeting Sp1 and survivin [36]. We hypothesized that TA effects may be evident in undifferentiated and actively growing (malignant) cells, but may not be toxic to differentiated cells.…”

“…Pre-clinical studies from our laboratory and others demonstrated the anti-cancer and chemopreventive response of tolfenamic acid (TA, 2-(4-chloro-3-methylanilino)benzoic acid) in a variety of human cancers. TA has been studied in pre-clinical models for adult (pancreatic [26,27], lung [28], esophageal [14,29], ovarian [30], prostate [31,32], and colon [33,34]) cancers and pediatric (leukemia [35], neuroblastoma [36], and medulloblastoma [37]) malignancies. Experimental results from our laboratory also showed a chemopreventive effect by this drug in an animal model of esophageal cancer [29,38].…”

Reactive oxygen species mediate tolfenamic acid-induced apoptosis in human colorectal cancer cells

“…Tolfenamic acid (TA) is a (N-(2-methyl-3-chlorophenyl)-anthranilic acid and possesses anti-cancer activity in several cancers such as pancreatic cancer [13], colorectal cancer [14][15][16], lung cancer [17], breast cancer [18], ovarian cancer [19], prostate cancer [20,21], brain cancer [22,23], esophageal cancer [24], sarcoma [25] and head and neck cancer [26]. Anti-cancer effect by TA is influenced by expressions of various cancer-related genes and cell signaling pathways.…”