Sagar Shelake

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

Abstract 1532: Mithramycin induces the antiproliferative activity of chemotherapeutic agents in Ewing sarcoma cells

Ewing sarcoma (ES) is one of the most frequent primary bone and soft tissue tumors that occur in pediatric and adolescent population. ES is often diagnosed after the disease has already metastasized. With available treatment options, the prognosis of metastatic ES patients is poor and the 5-year survival rate is less than 20%. Therefore, identifying precise targeted therapies to induce therapeutic response in ES patients is urgently needed. An oncogenic fusion protein and transcription factor EWS-FLI1 is associated with more than 85% of ES tumors. Mithramycin has been identified as an effective agent to target EWS-FLI1 and is currently in clinical trials. In this study, we investigated the efficacy of Mithramycin to induce the anti-proliferative activity of chemotherapeutic agents commonly used for the treatment of this malignancy using human ES cell lines. TC71, TC32, CHLA32, CHLA10 and TC205 cells were treated with increasing concentrations of Mithramycin or Vincristine or Doxorubicin or Etoposide. Cell growth inhibition was evaluated at 24 and 48 h post-treatment using CellTiter Glo kit. Results showed a dose/time-dependent anti-proliferative effect for all agents. To confirm the effect of Mithramycin on selected cell lines, mRNA expression of downstream targets of EWS-FLI1 (ID2, LDB2, NROB1 and RCOR1 genes) was determined by quantitative PCR following treatment with Mithramycin. Mithramycin significantly down-regulated all tested genes and these results are in agreement with published work. In order to determine the combination response, cell growth inhibition of the chemotherapeutic drugs was assessed in the presence of added Mithramycin. The combination Index was evaluated by Chou-Talalay method. Further studies were performed for the combination of Mithramycin and Vincristine to investigate its effect on apoptosis and cell cycle arrest using CHLA10 and TC205 cells. Apoptotic markers such as the expression of cleaved Poly (ADP-ribose) polymerase (c-PARP) and survivin were determined by Western blot analysis. The apoptotic cell population was measured by Annexin-V staining using flow cytometry. When compared to individual agents, the combination of Mithramycin and Vincristine showed greater effect on apoptosis in both cell lines as evinced by an increase in Annexin-V positive cells, decrease in survivin expression and up-regulation of c-PARP. Overall, these results indicate the potential clinical advantage of combination treatment involving Mithramycin along with other chemotherapeutic agents for inhibiting the growth of ES cells. Molecular profiling analysis is underway to elucidate the candidate pathways associated with beneficial effect of this combination treatment. Note: This abstract was not presented at the meeting. Citation Format: Anish Ray, Bhavani Nagarajan, Umesh T. Sankpal, Sagar Shelake, W. Paul Bowman, Andras Lacko, Riyaz M. Basha. Mithramycin induces the antiproliferative activity of chemotherapeutic agents in Ewing sarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1532. doi:10.1158/1538-7445.AM2017-1532

Abstract 4818: Tolfenamic acid and curcumin treatment induces pancreatic cancer cell growth inhibition via suppressing Sp1 expression, NF-kB translocation to nucleus

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-a 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.

Abstract 2467: Association of specificity protein 1 and survivin expression in medulloblastoma: Identifying effective therapeutic targets

Medulloblastoma (MB) is an aggressive malignant brain tumor diagnosed mostly in children. MB is typically treated using a multimodal approach consisting of surgery, craniospinal irradiation, and chemotherapy. These treatments cause delayed consequences in pediatric patients. In order to treat this malignancy effectively, it is important to identify critical markers associated with the disease and finding specific agents to target such markers. The transcription factor, Specificity protein 1 (Sp1) and an inhibitor of apoptosis protein, survivin are over expressed in many cancers and associated with poor prognosis. Sp1 mediates the expression of several oncogenes including survivin. Even though some evidence exists for the expression of survivin, the information on Sp1 is still limited in MB. The primary objective of this study was to determine the association of Sp1 and survivin in MB and developing the strategies to target these candidates using less toxic compounds. A human MB tumor tissue array consisting of 20 tumor and 3 normal controls was stained for Sp1 and survivin using specific antibodies. Tumor specimens showed distinct expression for both Sp1 and survivin in majority of tumor tissues. Using small interference RNA (siRNA) technology, the expression of Sp1 and survivin was inhibited in MB cell line, DAOY, and the cell viability was determined at 24 and 48 h using CellTiter-Glo kit. We observed that the inhibition of Sp1 and survivin by siRNA correlated with a decrease in DAOY cell viability. Previously, our laboratory showed that a small molecule, Tolfenamic Acid (TA) inhibits cell proliferation and tumor growth in mice via targeting Sp1 and anti-apoptotic protein, survivin. Taken together these results highlight the significance of targeting Sp1 and survivin for inhibiting MB cell proliferation and tumor growth in mouse model. Survivin has also been demonstrated to play a role in chemoresistance. We therefore, tested the combination treatment(s) involving TA and standard chemotherapeutic agents, Vincristine (Vinc) and Cisplatin (Cis) using MB cell lines, DAOY and D283. Cells were treated with TA, Vinc, Cis and combination of TA+Vinc and TA+Cis and the effect on cell viability and apoptosis was determined. Each drug alone caused a time and dose dependent decrease in cell viability that was enhanced in the presence of TA. The combination treatment also resulted in a 2-3 fold increase in apoptosis as determined by annexin V and propidium iodide staining. It is plausible that targeting Sp1 and/or survivin induces the susceptibility of MB cells to chemotherapeutic drugs. These preliminary results demonstrate the efficacy of combining inhibitors of Sp1 and survivin with chemotherapeutic agents to enhance their therapeutic response while limiting the toxicity and side-effects. Citation Format: Umesh T. Sankpal, Don Eslin, W. Paul Bowman, Jeffrey C. Murray, Irene Sanchez, Michelle Jones, Sagar Shelake, Yazmin Hernandez, Anmol Wadwani, Hassaan Patel, Ashni Dudhia, Riyaz M. Basha. Association of specificity protein 1 and survivin expression in medulloblastoma: Identifying effective therapeutic targets. [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 2467.