Combination Therapy to Enhance Anti-Cancer Activity Against Neuroblastoma and Ewing Sarcoma Cells

Anti-neoplastic drugs used for the treatment of various cancers often cause severe toxicity. Chemotherapy induced side effects are highly detrimental in pediatric and adolescent and young adult (AYA) cancers. This project is aimed at evaluating the strategies to induce the therapeutic efficacy of anti-neoplastic agents in a pediatric and an AYA cancer models, neuroblastoma (NB) and Ewing Sarcoma (ES) respectively. NB is a most common type of pediatric cancer that arises from autonomic nervous system while ES is a very aggressive bone and soft tissue sarcoma diagnosed in AYA patients. Small molecules such as Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are under investigation for their anti-cancer activities. The transcription factor, Specificity protein 1 (Sp1) and an inhibitor of apoptosis protein, survivin are over-expressed in multiple cancers including NB and Sarcoma. Sp1 and survivin has been shown to be associated with poor prognosis and resistance to chemo-/radiation therapy in some cancer models. Tolfenamic acid (TA), a NSAID has been shown to induce the anti-proliferative activity of several cancer cells by downregulating Sp1 and survivin. Therefore, we hypothesized that targeting these candidates using TA will enhance the anti-proliferative activity of anti-neoplastic agents in NB and ES cells. After initial screening of anti-neoplastic agents using NB and ES cell lines, cis-retinoic acid (RA) was chosen for NB and vincristine (Vin) was selected for ES. Experiments were conducted using the NB and ES cell lines to assess the individual and combination treatment of TA and anti-neoplastic agents (RA and Vin) and the results presented below in 3 specific aims. Aim 1: Chemotherapy used for the treatment of NB often causes long-term side effects in pediatric patients. Moreover, resistance to chemotherapy and disease relapse is serious concerns in high-risk neuroblastoma (HRNB). RA is used as an anti-neoplastic agent to induce remission in HRNB. However, HRNB patients treated with RA suffers from severe toxicities and the relapse rate is very high. Therefore, it is important to develop alternative strategies for effective treatment of HRNB. In this aim we investigated the efficacy of TA, for enhancing the anti-proliferative effect of RA in NB cell lines. TA and RA combination treatment resulted in decreased Sp1 and survivin expression which was accompanied by decreased cell growth and increase in apoptosis. This study demonstrated that the TA-mediated inhibition of Sp1 in combination with RA provides a novel therapeutic strategy for the effective inhibition of HRNB cells. Aim 2: In this aim, we demonstrated a strategy to target Sp1 and survivin using TA to inhibit ES cell growth. Our results revealed that TA inhibited cell viability, induced G0/G1 cell cycle arrest and increased apoptosis in ES cells. Mechanistically, we showed that that TA inhibited Sp1 and survivin protein and/or mRNA expression, disrupted Sp1 DNA-binding and inhibited ES cell proliferation. The results of this investigation suggest that targeting Sp1 and survivin could be an effective strategy for inhibiting ES cell growth. Aim 3: Vincristine, a plant derived alkaloid, is an integral part of chemotherapeutic regimens used for the treatment of ES. Vin treatment is known to cause severe long-term side effects such as sensory and motor neuropathy. Therefore, there is a need to identify novel strategies to improve the efficacy and reduce toxicities associated with the use of Vin. We showed that TA enhanced anti-cancer activity of Vin in ES cells. Specifically, TA and Vin combination treatment decreased Sp1 and survivin protein expression which was accompanied by an increase in apoptotic markers and cell cycle (G2/M) arrest, thereby leading to ES cell growth inhibition. Results of this study demonstrated that TA could enhance the anti-proliferative activity of Vin and suggesting that TA+Vin combination could be more effective against ES cell growth. Taken together, these data suggest that combination of TA and anti-neoplastic agent(s) treatment could be developed as a novel and safer therapeutic strategy for cancer treatment by targeting Sp1 and survivin.