Combination of clotam and vincristine enhances anti-proliferative effect in medulloblastoma cells

Highlights

• Combination of clotam and vincristine induces anti-cancer activity in medulloblastoma cells.

• Clotam and vincristine combination indiscriminately inhibiting SHH and 4/3 sub group cell lines

• Targeting survivin with clotam is effective for sensitizing medulloblastoma cells to chemotherapy.

• Clotam potentially destabilizing the microtubules for inducing the growth arrest caused by vincristine

Abstract

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.

Introduction

Medulloblastoma (MB) is an embryonal tumor of the posterior fossa which is located in cerebellum and has a propensity to disseminate throughout the central nervous system (Louis et al., 2016). Each year, 400 to 500 children in the United States are diagnosed with MB, with more frequent occurrences in males than females with a ratio of approximately 2:1 (Polkinghorn and Tarbell, 2007; Gopalakrishnan et al., 2015). Moreover, the overall survival rate in infants is lower (between 30 and 50%) than in older children as their disease tends to be more aggressive with macroscopic metastatic features (M2/M3) (Gottardo and Gajjar, 2006). Currently, a multimodal approach is applied as the standard treatment strategy for MB. Current standard of care therapy includes gross total resection, craniospinal irradiation and adjuvant chemotherapy (Bourdeaut et al., 2011; Pollack, 2011). Chemotherapy is the preferred option over radiation for children below the age of three years. Vincristine, lomustine, cisplatin, methotrexate and temozolomide are some of the common chemotherapeutic drugs used in treatment regimens of MB (Othman et al., 2014). However, despite good outcomes after treatment many survivors have significant medical problems including neurological, neurocognitive and neuroendocrine sequela that affect their quality of life (Ribi et al., 2005). In this current scenario of chemotherapeutic clinical outcome, it is important to give emphasis on enhancing the efficacy of the present anti-cancer drug treatments while reducing long-term side effects.

Non-steroidal anti-inflammatory drugs (NSAIDs) are known for their anti-inflammatory, anti-pyretic and analgesic properties. Their role as anti-cancer and chemopreventive agents have also been well established via numerous experimental, clinical and epidemiological studies (Rayburn et al., 2009). They promote anti-neoplastic activity by targeting the proliferative, angiogenic and metastatic processes of cancer cells (Liggett et al., 2014; Hilovska et al., 2015). Inhibition of prostaglandin biosynthesis from arachidonic acid by blocking the activities of cyclooxygenase (COX)-1 and COX-2 is the conventional mechanism of NSAIDs (Vane and Botting, 1997; Smith et al., 2000). However, this mechanism has undesirable consequences including gastric bleeding, cardio-toxicity and kidney failure (Thun et al., 2002; Khan and Fraser, 2012). Side effects associated with the COX-dependent mechanism have stimulated research into exploring the various COX-independent enzymatic pathways of NSAIDs (Liggett et al., 2014). Evaluating NSAIDs with lower or negligible toxicities that may modulate specific cellular and molecular targets applicable to cancer therapeutics has become a growing field. One such COX-independent NSAID which has been extensively studied is tolfenamic acid (TA). TA is commercially known as Clotam® or Tufnil® and is used for treating migraine headaches in Europe and Asia (Vaitkus and Pauza, 2002) and known to cross blood brain barrier (Subaiea et al., 2011). Compared to other NSAIDs, TA is known to have a low gastro-ulcerogenicity profile (Eskerod, 1994). Interestingly, Our laboratory and others have investigated the anti-tumorigenic activity of TA in pre-clinical models of several different types of malignant cancers, including the pediatric cancers leukemia, neuroblastoma and MB (Abdelrahim et al., 2006; Sankpal et al., 2012; Eslin et al., 2013a; Eslin et al., 2013b; Sutphin et al., 2014). The proposed mechanism for its anti-cancer activity is inhibiting the expression of Specificity protein transcription factor 1 (Sp1) and survivin, an inhibitor of apoptosis protein (IAP) (Basha et al., 2011; Shelake et al., 2017). However, in this investigation, the focus is on survivin due to its association in resistance to chemo-or radiation therapy. Survivin is known for its dual role in inhibiting apoptosis and promoting mitosis. Survivin has a prognostic importance due to its elevated expression in several metastatic cancer types (Ryan et al., 2009). Clinical findings have also associated overexpression of survivin as poor prognosis determinant in MB (Abdel-Aziz et al., 2013). In addition, evidence indicates that TA can regulate the expression of survivin, suggesting it as a viable option in attenuating overexpression of survivin in various tumors (Konduri et al., 2009; Chen et al., 2011). We have previously shown the downregulation of survivin expression by TA in MB cell lines and animal xenograft models lead to tumor growth inhibition (Eslin et al., 2013a).

Understanding the mechanisms by which NSAIDs have an anticancer effect has helped in understanding how to incorporate these medications in the therapy. There are numerous studies, both preclinical and clinical, that have demonstrated the use of such anti-inflammatory and analgesic agents as effective adjuvants for conventional chemotherapies (de Groot et al., 2007). Identifying, which standard of care chemotherapy medications to use in conjunction with anticancer NSAIDs has the potential to show increased efficacy and possibly decreased long-term side effects. Vincristine® (VCR), a commonly used chemotherapeutic agent for MB treatment, is known to induce neurotoxic effects in children such as peripheral neuropathies and seizures (von Bueren et al., 2009). We have undertaken this study to see whether or not the addition of TA with VCR could lead to increased efficacy of VCR in MB. In this study, we used human MB cell lines (DAOY and D283) to investigate the combination effect of TA and VCR to achieve an enhanced anti-tumorigenic effect. We hypothesize that the addition of TA sensitizes MB cells to the chemotherapeutic drug VCR by targeting survivin expression.