Cancer
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21618
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Browsing Cancer by Author "Basha, Riyaz"
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Item Anti-Proliferative Effect of Copper Tolfenamic Acid in Neuroblastoma Cell Lines(2019-03-05) Basha, Riyaz; Sankpal, Umesh; Maram, Rajasekhar; Grebennikov, SarahAnti-Proliferative Effect of Copper Tolfenamic Acid in Neuroblastoma Cell Lines Sarah Grebennikov, Yazmin Hernandez, Rajasekhar Maram, Umesh T. Sankpal, and Riyaz Basha Background:Neuroblastoma (NB) is a neuroendocrine tumor of the sympathetic nervous system most commonly found in the adrenal medulla. It is the most common extracranial tumor in infants with an average age of onset of 1 year. While presentation in children over the age of 5 is rare, the prognosis is markedly worse due to the higher likelihood of an aggressive malignancy with metastasis to lymph nodes and bone marrow. Current treatment modalities include surgical resection, chemotherapy, radiotherapy, and autologous stem cell transplant. These treatments are highly efficacious, however there are several associated side effects. Specifically, chemotherapeutic side effects are dose dependent and can range from mild stomach pain to more severe and serious complications including hearing loss, myelosuppression, and neurotoxicity. To limit these side effects, we are investigating anti-cancer agents with limited side-effects. Copper Tolfenamic Acid (Cu-TA) is metallic complex of an anti-cancer Non-Steroidal Anti-inflammatory Drug, Tolfenamic acid which is known to inhibit anti-apoptotic protein, Survivin and inhibits cancer cell growth. Hypothesis: We hypothesize that Cu-TA down-regulates survivin and inhibits neuroblastoma cell growth more effectively than TA. Methods: NB cell lines, SMS-KCNR and LA155n cell lines (from ATCC) were treated with increasing concentrations of Cu-TA or TA (0, 5, 10, 20, 40 and 80 µM). CellTiter-Glo reagent was added to the 96-well plate, and readings were taken at 24 and 48 hours. Using this data, IC50 values were calculated using SigmaPlot software. The effect of Cu-TA on Survivin protein expression was measured using western blot analysis. Results: Cell viability data showed a dose dependent decrease due to Cu-TA treatment in both cell lines. Analysis of the Western Blot confirms that there was a decrease in the survivin protein in the cells treated with Cu-TA. Conclusion:These results demonstrate that Cu-TA is an inhibitor of survivin and more effective at inhibiting NB cancer cells than TA alone. Since survivin is associated with resistance to chemotherapy, if Cu-TA sensitizes NB cells to chemotherapy, it will help reduce the side effects of chemotherapy while maintaining the efficacy of treatment.Item Diffuse Cutaneous Mastocytosis and its potential comorbidities in pediatric patients – a case study.(2019-03-05) Basha, Riyaz; Bowman, Paul; Hamby, Tyler; Smith, JohnBackground: Mastocytosis is the pathologic proliferation and accumulation of mast cells in various tissues of the body. There are different forms of mastocytosis that can present in pediatric patients including systemic (SM), cutaneous (CM) and diffuse cutaneous mastocytosis (DCM). Both the CM and DCM forms have the potential to progress into SM as the patient reaches adulthood. Mastocytosis has been shown to be comorbid with joint pathologies including Ehlers-Danlos syndrome and inflammatory gastrointestinal conditions such as eosinophilic esophagitis. The greatest risk among patients with mastocytosis is anaphylaxis. Case information:A13-week-old male presented to his primary care physician with erythematous spots on his torso and arms, and was diagnosed with eczema. The spots grew and transformed morphologically over the next month and a referral to dermatology was made. Upon biopsy of the original lesion (on the torso), the diagnosis of DCM was made. Over the coming months, symptoms progressed and comorbidities—including joint hypermobility (diagnosed with Ehlers-Danlos syndrome), dysphagia and diarrhea—arose. The patient broke his distal radius while crawling, due to his mast cell disorder and severe vitamin D deficiency. Conclusions: In most children with DCM, symptoms will partially or fully resolve by adolescence. But for some patients, the disease can progress to SM. Numerous comorbidities can occur, as did in this case. Current treatment strategies are wide ranging, from topical glucocorticoids to specialized UV radiating therapy. The specific approaches to this disease are still being understood, with recent investigations into immunological treatment modalities. The individuality of each case is crucial for health care professionals to recognize.Item Exploring Less Toxic Combination Treatment Options for Inducing Anti-Cancer Activity in Medulloblastoma Cells(2019-03-05) Smith, Kolton; Grebennikov, Sarah; Sankpal, Umesh; Bowman, W.; Basha, Riyaz; Schullek, MelissaPurpose.Medulloblastoma (MB) is the most common type of malignant pediatric brain cancer and is typically located in the posterior fossa. There are four subgroups within MB: Wnt, Sonic-Hedgehog, Group 3, and Group 4. Due to differences in pathology, signaling pathways, and gene expression, each subgroup is approached differently with respect to treatment, based upon differences in prognosis. Currently, the standard treatment approaches include surgical resection, radiotherapy, and chemotherapeutic agents such as etoposide, vincristine, and cisplatin. Survivors often suffer from severe long-term side effects including neurocognitive deficits and the potential for a future second neoplasm due to the tumorigenic potential of aggressive combination therapies. Because of these side-effects, there is an urgent need for effective and less toxic therapeutic strategies for the treatment of MB. Through prior research we have demonstrated the combination of etoposide alongside less toxic anti-cancer agents potentially increases anti-cancer activity in Ewing Sarcoma. We hypothesize that using a combination of etoposide with other sensitizing agents can also enhance the anti-cancer activity in MB cell lines. Methods. DAOY cells were cultured with increasing concentrations of Etoposide (ETO), Mithramycin-A (MIT), BNS-22 and Tolfenamic acid (TA) and the cell growth was monitored at 48 hours using CellTiter-Glo kit (luminescence cell viability assay). Dose-curves were then generated using sigma-plot software. After calculating the IC50 values for each agent, low dose of ETO (half of IC50 value) and IC50 value of other agents were tested for the combination treatment. Results. Overall, we observed decreased cell viability in a dose and time dependent manner for all tested agents. The IC50 values derived from the dose curves were 1 µg/ml for ETO, 33.3 nM for MIT, 15 µg/ml for TA, and 14.5 µM for BNS. The combination treatment using 0.5 µg/ml ETO and other agents (IC50 values) showed cell growth inhibition greater than any single agent in DAOY cells. The analysis revealed that the combination of ETO (0.5 µg/ml) plus BNS-22 was very effective. Conclusions: These preliminary data demonstrate promise in creating combination therapy of ETO with BNS-22 to treat DAOY cell lines. For better applications, similar experiments should be done with more cell lines representing various sub-groups of MB and to be confirmed by in vivoassays.Item Metformin inhibits medulloblastoma cell growth and increases sensitivity to chemotherapy drugs(2019-03-05) Basha, Riyaz; Bowman, Paul; Sankpal, Umesh; Hong, JuliePurpose: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Standard treatment is chemotherapy and radiation, both of which can be associated with long-term toxicity for pediatric patients. This project is focused on the use of metformin in the treatment of medulloblastoma. Metformin (MET) is an anti-diabetic drug with low toxicity that has been shown to have anti-cancer properties. We hypothesize that MET will inhibit MB cell growth and enhance the effect of chemotherapy and anti-cancer agents such as vincristine (VCR) and valproic acid (VPA) when used in combination, possibly by inhibiting the expression of survivin protein. Methods: MB cells (DAOY) were treated with increasing doses of MET (1-30 mM), VCR (1-16 nM), and VPA (1-30 mM). For combination treatment, DAOY cells were treated with selected doses of VCR (1, 2, 4 nM) and VPA (0.9, 1.8, 3.5 mM) alone or in the presence of MET (10 and 20 mM). Cell viability was assessed at 48 h post-treatment using the CellTiter-Glo cell viability assay kit. For western blot analysis, DAOY cells were treated with increasing doses of MET (0, 5, 10, 20 mM) for 24 and 48 h. Cells were harvested and protein extracts were prepared and used for determining survivin expression. Results: Treatment with MET, VCR, and VPA alone resulted in a decrease in cell viability in a dose and time dependent manner. The combination of MET+VCR resulted in greater inhibition of cell proliferation with 78.99% inhibition in comparison to MET alone (51.5%) or VCR alone (46.02%). The combination of MET+VPA resulted in greater inhibition of cell proliferation with 84.88% inhibition in comparison to MET alone (52.6%) or VPA alone (47.81%). Western blot analysis of MET treated cells showed a dose and time dependent decrease in survivin expression. Conclusion: Our experiments demonstrate the potential of MET as a novel therapeutic agent for the treatment of MB based on its ability to inhibit proliferation and enhance the activity of anti-cancer agents. These results also suggest that MET’s effect could be partially mediated by the down-regulation of survivin, a protein known to be involved in the inhibition of apoptosis and resistance of cells to chemotherapy. The low toxicity of metformin and its ability to sensitize medulloblastoma cells could potentially result in lowering chemotherapy associated toxicities, leading to improved quality of life for long-term survivors.Item Survivin as a New Target for Neuroblastoma(2019-03-05) Basha, Riyaz; Umesh, Sankpal; Hernandez, Yazmin; Zakhary, EmilyBackground: Neuroblastoma is the most common solid peripheral nervous system pediatric tumor found in pediatrics. Neuroblastoma (NB) commonly metastasizes through the lymphatic system and bone marrow, and has a particularly poor prognosis in children older than 18 months, with five-year survival rates typically around 40-50%. Our group is investigating for less toxic agents against NB cells. We found that Tolfenamic acid (TA), a small molecule had anti-cancer effects in high risk neuroblastoma (HRNB) cell lines. Survivin has been associated with poor prognosis in several types of cancers. Survivin is a protein that specifically inhibits the caspase apoptotic proteins thus negatively regulating apoptosis. We conducted a search of various databases in order to investigate the association of Survivin with the survival of NB patients. The other objective was to test an agent that could target Survivin an inhibit NB cell growth. Materials: We utilized R2 genomics and visualization platform to generate survival curves, looking specifically at overall and relapse-free survival probabilities in NB patients. The graphs were made using the data from 88 patients. We also used NB cell line for in vitro testing. SH-SY 5Y cells were cultured it in the presence of copper TA and cell viability was assessed at 24 and 48 hours. Protein extracts were prepared and analyzed for the expression of Survivin using Western blot analysis. Results: We found that the overall survival probability for NB patients with high expression of Survivin had a significantly poorer prognosis (p: 8.5e-9), than those with lower expression of Survivin. Similarly, the relapse-free survival probability curve also demonstrated that high expression of Survivin was associated with a poorer prognosis (p: 1.9e-6) than patients who had lower expression levels. Thus demonstrating that there is a strong association with high Survivin expression and poor prognosis. We also found that Cu-TA acted as an effective inhibitor to Survivin in our laboratory research with the SHSY-5Y cell line. Conclusion: The survival curves showed a strong association of Survivin with poor prognosis. The complexed agent, Cu-TA acts as an efficacious inhibitor of the HRNB cell line SHSY-5Y potentially inhibiting Survivin. Going forward, further research should be done to identify novel less toxic therapeutic agents to target Survivin, in hopes of better treating HRNB cell lines.Item The role of Specificity Protein 1 and downstream target Survivin in Ovarian Adenocarcinoma Survival and Prognosis(2019-03-05) Nash, Madeline; Chandra, Ashwin; Nair, Maya; Vishwantha, Jamboor; Basha, Riyaz; Lin, ChristinePurpose: Ovarian adenocarcinoma is a solid proliferation of ovarian glandular epithelium. These are the most threatening types of gynecologic malignancies. Although many risk factors are involved in determining prognosis, the study of germline mutations related to tumorigenesis is the focus of this study. It is well established that Specificity protein 1 (Sp1), a transcription factor regulates key genes associated with cancer including survivin (baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5). Survivin is an anti-apoptotic protein which is known to be associated with poor prognosis in multiple cancers and displays therapy-resistant tumor characteristics. Sp1 has also been associated with poor prognosis in several different types of cancers. The role of these two proteins in the development of ovarian adenocarcinoma and their impact on prognosis remains unclear. Methods: The R2 genomics visualization platform was used to generate Kaplan-Meier curves for survival probabilities in patients with ovarian adenocarcinoma who express high and low levels of Sp1 and Survivin. Both curves were generated using data from 107 patients. There were 38 patients in the high Sp1 expression group and 69 patients in the low Sp1 expression group. In the Survivin (BIRC5) group there were 70 patients with high expression and 37 patients in the low expression group. Results: The survival curves show that patients with ovarian adenocarcinoma who express higher levels of Sp1 had a significantly poorer prognosis compared to patients with lower levels of Sp1 expression (p = 0.023). Similarly, patients with high levels of survivin protein expression were found to have a significant decrease in overall survival probability compared to patients who had low levels of survivin expression (p = 0.049). Conclusion: There is a strong association with high expression of either Sp1 or survivin with poor prognosis in patients with ovarian adenocarcinoma. Moving forward, studies are in development to elucidate the roles that Sp1 and survivin play, individually and together, in the evolution of ovarian adenocarcinoma. We are also interested in exploring the relationship of differential expressivity of Sp1 and survivin in association with age, race and ethnicity among patients with ovarian cancer.Item Trial of Pazopanib in a Multiply Relapsed Osteosarcoma Patient(2019-03-05) Albritton, Karen; Akers, Lauren; Ray, Anish; Basha, Riyaz; Elete, KunalBackground: Osteosarcomas (OS) are typically found among adolescents and young adults and usually affect the long bones around the knee. The current treatment options for relapsed OS include surgery, chemotherapy, targeted therapy, or some combination of these modalities. Constitutive activation of tyrosine kinase mediated pathways leading to up-regulation of cell division and growth have been implicated in OS. This study identifies a patient at Cook Children’s Medical Center (CCMC) who, upon relapse, was treated with pazopanib, a multi-tyrosine kinase inhibitor, on compassionate basis, which led to stability of disease, along with treatment related toxicities. Case Information: A 25-year-old female initially presented with left femur osteosarcoma and was treated with chemotherapy consisting of methotrexate, doxorubicin and cisplatin followed by amputation and neo-adjuvant chemotherapy. Following 6 years of remission, she presented with two lung masses that were resected along with chemotherapy and radiation. After almost 2.5 years, she presented with progressive tumor in the right pleural base for which she was treated with pazopanib. She demonstrated positive response with stable size of tumor and increased homogeneity (suggestive of tumor necrosis) but ended treatment after 3 months due to hypothyroidism and GI toxicity, namely diarrhea. Within just 1 week of discontinuation, she had a concerning increase of 17% in her lesion. Thereafter, she has relapsed multiply but remains alive 18 months after discontinuing pazopanib. Conclusion: Despite the positive response seen to pazopanib, it’s toxicity profile can be over bearing for patients. In a retrospective analysis by Velho et al, a study involving 113 patients treated with pazopanib resulted in about 12% of those discontinuing treatment due to fatigue, diarrhea, and nausea/vomiting. In another study, described by Umeda et al, 3 patients with relapsed osteosarcoma who were treated with pazopanib were all alive at 21 months or longer. Of those, two discontinued treatment despite positive response due to nausea/fatigue, lymphopenia, anemia, hypothyroidism, and elevated alkaline phosphatase. Our experience as well of those as others suggests that pazopanib may have a role in prolonging survival among patients with osteosarcoma, however the extent of the side effects has clearly contributed to a lesser than optimal length of treatment.Item Understanding the Mechanism of Action of Copper-Tolfenamic Acid’s Anti-cancer Activity in Pancreatic Cancer Cells(2019-03-05) Prokai, Laszlo; Umesh, Sankpal; Basha, Riyaz; Hurtado, Myrna; Levesque, BlairPurpose: Non-steroidal anti-inflammatory (NSAID) agents have been proven to have anti-cancer activity. Our group is investigating the use of NSAIDs as sensitizing agents to use alongside standard chemotherapy. This combination increases the efficacy of chemotherapy to accomplish higher anti-cancer activity at relatively low doses, thereby reducing the side effects. It is desirable to find anti-cancer NSAIDs with low Inhibitory concentration (IC50) values. The NSAID, tolfenamic acid (TA), has been tested in preclinical studies for anti-cancer activity against pancreatic cancer (PaCa). Recently, it is shown that metal complexes of NSAIDs enhances the efficacy. Methods: We investigated the anti-proliferative activity of copper-tolfenamic acid (Cu-TA) using 12 cancer cells and reported that the Cu-TA complex had a stronger therapeutic response and lower IC50 values by 30-80% compared to TA. The goal of this investigation is to determine the mechanism by which Cu-TA induces anti-cancer activity in PaCa cell lines. MIAPaCa2 cells were treated with vehicle or Cu-TA (IC50 value) and processed by Next Generation Sequencing (NGS). Ingenuity Pathway Analysis was used to determine the functional significance of the altered gene expression. The top upstream regulators were confirmed by Western blot analysis. Results: Several networks, regulators, and molecular and cellular functions were found to be affected by the Cu-TA treatment. qPCR and Western blot analysis were used to assess and confirm the alterations in the expression of the candidate markers in PaCa cells. Previously, confirmatory studies were performed using MIAPaCa2 cells. Due to the heterogeneity of PaCa, in this study we used a second cell line PANC1 for similar experiments. Tumor protein p53, human epidermal receptor growth factor 2, Specificity protein 1 and signal transducer and activator of transcription 3 were the top upstream regulators confirmed by Western blot analysis. It was demonstrated by qPCR of selected genes, Centromere protein F, DNA damage inducible transcript 3 and S-phase kinase associated protein 2 that Cu-TA is efficacious at a lower dose than TA. Conclusion: NGS and Ingenuity Pathway analysis identified important pathways and genes effected by Cu-TA. In this investigation, PANC1 showed similar results as MIAPaCa2 cells. The genes and pathways that were altered by treatment with Cu-TA involved cell survival or apoptosis demonstrating that Cu-TA is modulating genes associated with cancer. This identifies the potential of Cu-TA as an effective anti-cancer agent.