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. Anti-cancer NSAID, Tolfenamic acid (TA) and Cur combination was evaluated using PC cell lines. L3.6pl and MIA PaCa-2 cells were treated with Cur (5–25 μM) or TA (25–100 μM) or combination of Cur (7.5 μM) and TA (50 μM). Cell viability was measured at 24–72 h post-treatment using CellTiter-Glo kit. While both agents showed a steady/consistent effect, Cur+TA caused higher growth inhibition. Anti-proliferative 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) were 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 augmented 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.
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.
Background: Clostridioides difficile infections have become more frequently diagnosed and associated with greater disease severity, which has resulted in an increase burden on the healthcare system. These increases are attributed to the increased prevalence of hypervirulent strains encompassing select ribotypes. These epidemic ribotypes were characterized as hypervirulent due to higher in vitro spore and toxin production, as well as increased incidence, severity and mortality within patients. However, it is unclear whether epidemic ribotypes are truly more virulent than non-epidemic ribotypes in vivo. Furthermore, there is conflicting evidence about the ability of a strain's in vitro phenotype to be predictive of their in vivo virulence. The goals of the current studies were to determine if epidemic ribotypes are more virulent than other ribotypes in animal models, and whether the in vitro virulence phenotype of an isolate or ribotype predict in vivo virulence. Results: To determine if epidemic strains were truly more virulent than other non-epidemic strains, the in vivo virulence of 13 C. difficile isolates (7 non-epidemic and 6 epidemic ribotype isolates) were determined in murine and hamster models of CDI. The isolates of epidemic ribotype of C. difficile were found to be more virulent in both the murine and hamster models than non-epidemic isolates. In particular, the group of epidemic ribotypes of C. difficile had lower LD 50 values in hamsters. The increased severity of disease was associated with higher levels of Toxin A and Toxin B production found in fecal samples, but not numbers of organisms recovered. The isolates were further characterized for their in vitro virulence phenotypes, e.g. toxin production, growth rates, spore formation and adherence of spores to intestinal epithelial cell lines. Although there were higher levels of toxins produced and greater adherence for the group of epidemic ribotypes, the in vitro profiles of individual isolates were not always predictive of their in vivo virulence. Conclusions: Overall, the group of epidemic ribotypes of C. difficile were more virulent in vivo despite individual isolates having similar phenotypes to the non-epidemic isolates in vitro.
Pancreatic cancer (PC) is an aggressive malignancy. The current treatment options have limited response in addressing poor prognosis and low survival rate. Hence it is important to identify novel agents and strategies for effective treatment. Previously the combination of phytochemical, curcumin (Cur) and cyclooxygenase (COX) inhibitor celecoxib was tested for improving therapeutic efficacy in PC models. The objective of current study is to identify a combination treatment involving a low toxic small molecule and a phytochemical with anti-cancer properties to inhibit PC cell growth. Experiments were also conducted to understand potential mechanisms associated with this combination. We tested the combination of an anti-cancer non-steroidal anti-inflammatory drug (NSAID), Tolfenamic Acid (TA) and Cur using PC cell lines, L3.6 and MIA PaCa-2. Cells were treated with 5-25 μM of Cur or 25-100 μM of TA or combination of Cur (7.5 μM) and TA (50 μM). Effect on cell viability was measured at 24, 48 and 72 h post-treatment using CellTiter-Glo kit. While the two agents showed anti-proliferative effect, Cur and TA combination caused higher growth inhibition. The cell growth inhibition was compared with two COX inhibitors, ibuprofen and celecoxib and the cardiotoxicity was assessed using cordiomyocytes (H9C2). TA showed significantly less cytotoxicity in cardiomyocytes when compared to celecoxib. The expression of transcription factors, Specificity protein1 (Sp1) and NF-kB, and an inhibitor of apoptosis family protein, survivin, were determined by Western blot analysis. The expression of NF-kB, Sp1 and survivin was decreased by combination treatment. The levels of reactive oxygen species (ROS) were also measured in flowcytometer. To evaluate the effect of these agents on apoptosis, the activity of caspase 3/7 was measured with caspase-Glo kit; apoptotic cell population was evaluated by Annexin-V staining (flow cytometry); and c-PARP expression was determined by Western blot analysis. When compared to individual agents, the combination treatment caused a significant increase in ROS levels and apoptotic markers. L3.6 and MIA PaCa-2 cells were treated with TNF-á to induce NF-kB translocation from cytoplasm to nucleus and the effect of individual (TA or Cur) and combined treatment (TA+Cur) on NF-kB translocation from cytoplasm to nucleus was evaluated by immunofluorescence. When compared to individual agents, the combination treatment caused a significant decrease in NF-kB translocation to nucleus. Cell cycle phase distribution was measured using flow cytometry. The combination treatment showed mostly DNA synthesis phase arrest; however TA caused cell cycle arrest in early phase (G0/G1). These results demonstrate that combination of Cur and TA effectively inhibits PC cell growth via inducing apoptosis and modulating cell cycle phase distribution. Citation Format: Riyaz M. Basha, Sarah F. Connelly, Ganji Purnachandra, Umesh T. Sankpal, Hassaan Patel, Jamboor K. Vishwanatha, Sagar Shelake, Leslie Tabor-Simecka1, Mamoru Shoji, Jerry Simecka W. Simecka, Bassel El-Rayes. Tolfenamic acid and curcumin treatment induces pancreatic cancer cell growth inhibition via suppressing Sp1 expression, NF-kB translocation to nucleus. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4818.
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