Browsing by Author "Sankpal, Umesh"
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Item A Biological Approach in Exploring Breast Cancer Disparities(2021) Ali, Arkoon; Sankpal, UmeshPurpose: In the United States, healthcare disparities lie across a multitude of diseases including breast cancer. These disparities are caused by multiple different factors such as socioeconomic status, education, environment, biology, and others. To overcome the disparities, each factor needs to be addressed with research and development. In the US, breast cancer death rates have significantly declined since the start of 21st century. However, looking closer at breast cancer outcomes between races, a significant racial disparity is seen between white and black women. Research has found that black women are diagnosed with breast cancer at an earlier age and with the most aggressive triple negative tumors. These factors attribute to the differences in outcomes between the black and white populations. Studies have also identified various biological differences between the two populations that can potentially affect outcomes for breast cancer. Method: Genomic data from breast tumor was used to obtain differentially expressed genes between the two populations. These were evaluated as possible biological markers for racial disparity in breast cancer. Results: Genes were identified that were expressed at higher levels in tumor tissue from black women compared to white. This also correlated with the lower survival rates observed for black women. Conclusion: In addressing the disparities of healthcare and more specifically those seen in breast cancer, novel approaches are needed to reduce mortality and improve outcomes for the black population. These approaches need to include a combination of public health strategies along with biological methods and techniques to improve outcomes.Item A Small Molecule Derivative as a Targeting Agent for Sp1 and Survivin Effectively Suppresses Pancreatic Cancer Cell Growth(2017-03-14) Sankpal, Umesh; Mahammad, Shahela; Chhabra, Jaya; Brown, Deondra; Gurung, Raj; Holder, Alvin; Basha, Riyaz; Hurtado, Myrna MsBackground: Pancreatic cancer has one of the most fatal malignancies due to its poor prognosis. It currently has a one-year survival rate of 20%. Current standard forms of treatment contain a high level of toxicity, thus preventing an increase in dosage or frequency. This issue poses the need for more effective, yet less toxic agents for treatment. Tolfenamic acid (TA) is most commonly used to treat migraines but has recently been demonstrated to contain anti-cancer properties. It is known to downregulate the Specificity Protein (Sp) transcription factor, Sp1. Sp1 regulates several genes involved in cell proliferation and apoptosis, including survivin, an inhibitor of apoptosis protein. Interestingly, a recent discovery proposed that a copper(II) complex with TA as a ligand can result in higher therapeutic response; however its efficacy was not tested in gastro-intestinal cancers. Purpose: In this study, we assessed the therapeutic efficacy of a Cu(II)- containing complex of TA (Cu-TA) using human pancreatic cancer cell lines. Methods: MIA PaCa-2 and Panc1 cells were treated with increasing concentrations of DMSO (vehicle), equimolar CuCl2 (negative control), TA or Cu-TA and the cell viability was measured at 24 and 48 h post-treatment using CellTiter-Glo kit. CuTA was further tested for its effect on Sp1 and survivin expression by Western blot and quantitative PCR. The activation of apoptosis was determined by measuring the activity of effector caspases using the Caspase 3/7-Glo kit and the apoptotic cell population through flow cytometric analysis using Annexin-V staining. Cell cycle arrest was assessed by flow cytometry with propidium iodide staining. Results: While both TA and Cu-TA inhibited pancreatic cancer cell growth in a dose/time-dependent manner. Cu-TA was highly effective in inhibiting Sp1 and survivin protein expression and showed similar trend for inducing apoptotic markers and causing cell cycle arrest in G2/M phase. The results of qPCR demonstrated that the expression of survivin mRNA was significantly lower following both Cu-TA and TA treatment; however, the mRNA expression of Sp1 remained unchanged. This indicates that TA and Cu-TA could be affecting Sp1 by a similar mechanism. Conclusions: These results demonstrate that Cu-TA is more effective than TA and potentially useful for pancreatic cancer treatment after clinical testing. Studies to understand precise underlying mechanisms are currently under investigation.Item Anti-proliferative activity of clotam and copper-clotam against T-cell Acute lymphoblastic leukemia cell line CCRF-CEM(2020) Basha, Riyaz; Siraj, Sohail; Sankpal, Umesh; Polu, Rujula; Patel, KrishnaPurpose: Acute lymphoblastic leukemia (ALL) is the most common type of cancer in children younger than 5 years. Patients with ALL have bone marrow that produces immature white blood cells, which are unable to effectively fight infections. NSAIDs are common pain reliving agents that act through COX inhibition, which stops the production of prostaglandins. Clotam (Tolfenamic Acid/TA) is an NSAID that has anti-tumor proliferative effects. It works through targeting specificity protein (Sp) transcription factors that assist cancer cells in inhibiting apoptosis. Our objective is to test TA and copper-TA (Cu-TA), a derivative of TA, to induce an anti-leukemic response. Methods: The T-cell ALL cell line CCRF-CEM was obtained from the American Type Culture Collection (Manassas, VA) and cells were cultured as per the supplier's instructions. A cell viability assay was performed in which cells were plated in a 96-well plate and treated with increasing concentrations of TA and Cu-TA. After 48-hours, the cells were lysed, and the amount of ATP in the cells was measured using luminescence. Using this data, IC50 values were calculated. Results: The IC50 values showed both TA and Cu-TA had anti-cancer proliferative effects. Cu-TA was 15 times more potent than TA in its ability to kill CCRF-CEM cells. Conclusion: Our results demonstrate that Cu-TA is more effective than TA for killing CCRF-CEM cells. This study suggests better implications of Cu-TA in ALL therapy, if further tested using pre-clinical models.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 Anti-proliferative effects of a copper(II) complex with a thiosemicarbazone ligand against selected human cancer cells(2023) Fiadjoe, Hope; Lambring, Christoffer B.; Sankpal, Umesh; Alajroush, Duaa; Smith, Chloe; Anderson, Brittney; Mann, Novia; Beebe, Stephen; Holder, Alvin; Basha, RiyazPurpose: The frequent relapse and drug resistance associated with the current cancer chemotherapy treatments necessitate the development of alternative strategies. Thiosemicarbazones are a class of metal chelators that have been explored to treat diverse human diseases, including cancer. Copper, a crucial structural component for many significant enzymes and a key catalytic co-factor in redox processes, is being explored for several medical applications. Additionally, the anti-cancer activity of certain chemotherapeutic agents can be enhanced by the use of copper-containing complexes. This study aimed to evaluate the antiproliferative effects of a copper(II) complex with a thiosemicarbazone ligand (Cu-acetylethTSC or [Cu(acetylethTSC)Cl]Cl·0.25C2H5OH (where acetylethTSC = (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide)) against human cancer cell lines, viz., medulloblastoma (DAOY, D283), glioblastoma (LN-229), Ewing sarcoma (TC205, CHLA10), and acute lymphoblastic leukemia (CCRF-CEM, SUP-B15). Methods: These selected cell lines were cultured using standard protocols. Cell viability was measured using a Cell Titer-Glo kit at 48 h after treatment with various concentrations of Cu-acetylethTSC. Each treatment group and the controls were read in triplicates and the data were plotted as percentage cell viability versus concentration of the complex. Dose-response curves were generated based on the cell viability data obtained, and IC50 values were calculated. Cardiomyocytes (H9C2) were also cultured and used to test cytotoxicity in non-malignant cells. Results: Cell viability was inhibited in a dose-dependent manner in all the selected cancer cell lines whiles that of H9C2 was not significantly affected. Conclusion: This indicates that Cu-acetylethTSC was selective for malignant cells. Further studies are underway to understand the efficacy, protein targets, and underlying mechanisms of the role of Cu- acetylethTSC.Item Assessing the cytotoxicity of investigational agent for cancer therapy against non-malignant cells(2020) Patel, Krishna; Mukka, Lasya; Sankpal, Umesh; Basha, Riyaz; Siraj, SohailBackground: The treatment of cancer requires chemotherapy (ChT). Most of ChT agents exhibit unwanted side-effect and cause damage to healthy cells. Side effects from commonly used ChT agents are leaving pediatric cancer survivors with lasting damage to organ systems, specifically the heart. Studies conducted by our group demonstrated the anti-cancer activity of clotam (tolfenamic acid-TA) and copper-clotam (Cu-TA). Cu-TA is showing higher cytotoxicity against cancer cells even at much lower dose than TA in pancreatic cancer cells. Our long term objective is to test these agents to sensitize cancer cells to ChT. Methods: Cardiomyocytes H9c2 (cell line derived from rat heart tissue) originally obtained from the American Type Culture Collection (Manassas, VA) were cultured as per the supplier's instructions. H9c2 cells were treated with TA or Cu-TA or Doxorubicin and combinations (for example, TA and Doxorubicin) and cell viability assay was measured using CellTiter-Glo (Promega) kit at 48 hours post-treatment following manufacturer's instructions. Results & Conclusion: We found that TA or Cu-TA are not inducing toxicity in H9c2 cells at tested doses. TA kept more cells alive in conjunction with Doxorubicin than did the control. Our studies also show that H9c2 cells are not toxic to IC50 values of TA or Cu-TA determined with cancer cells. These results provide evidence that the tested investigational gents are not inducing toxicity in cardiomyocytes at tested doses and supports use of these agents in combination therapy with ChT.Item Carvedilol, an alternative for lowering liver stiffness in patients with cirrhosis and portal hypertension(2024-05) Guriginjakunta, Niharika; Sankpal, Umesh; Heck, Amber J.Liver cirrhosis, often associated with portal hypertension, presents a significant health burden globally. Carvedilol, a non-selective beta-blocker, has emerged as a promising therapeutic option for managing portal hypertension in patients with liver cirrhosis. This retrospective analysis assessed the effect of Carvedilol treatment on patients with liver cirrhosis and clinically suspected portal hypertension, focusing on its effects on liver function parameters, non-invasive fibrosis scores, and liver stiffness measurements. A total of 130 patients from the Liver Center of Texas were included in this retrospective analysis, comprising 65 patients in the treatment group receiving Carvedilol and 65 patients in the control group. Statistical analyses, including t-tests, were conducted to assess the differences between groups. Carvedilol treatment led to significant improvements in liver function parameters, including a reduction in AST levels, indicative of improved liver function. Non-invasive fibrosis scores, such as FIB-4, AGILE 3, AGILE 4, and APRI, showed notable improvements after Carvedilol treatment in the treatment group, suggesting a reduction in liver fibrosis and improved prognosis. Liver stiffness measurements using eKpa and CAP scores demonstrated significant reductions after Carvedilol treatment within the treatment group, indicating improved liver stiffness. The study suggests that Carvedilol is effective in managing portal hypertension in patients with liver cirrhosis. Further research is needed to confirm these findings in larger cohorts and evaluate the long-term efficacy, and safety of Carvedilol treatment. Additionally, addressing disparities in liver disease diagnosis and treatment is crucial for improving outcomes and reducing the burden of liver-related morbidity and mortality.Item Combination of Mithramycin and Standard Chemotherapeutic Agents Induces Anti-proliferative activity in Ewing Sarcoma cell lines(2018-03-14) Hunter, Abigail; Lout, Holly; Dunlap, Elissa; Sankpal, Umesh; Bowman, W. Paul; Basha, Riyaz; Ray, Anish; Albeer, LinaBackground/Hypothesis: Ewing sarcoma (ES) is a small, round, blue cell tumor found primarily in bones of adolescents. The EWS-FLI1 transcription factor is associated with proliferation of cancer cells and is over-expressed in [greater than] 85% of Ewing sarcoma cases. Mithramycin (MIT) is an antibiotic with antineoplastic properties and has been shown to inhibit EWS-FLI1. A recent trial of MIT treatment in ES patients found that hepatotoxicity precluded the administration of MIT at a dose required to inhibit EWS-FLI1 ([greater than]50nmol/L). We hypothesize that the efficacy of adjunct treatment can be enhanced if MIT is used along with standard chemotherapeutic agents such as Vincristine (VIN) and Etoposide (ETO). Combination treatment will reduce the effective dose of both Mithramycin and the standard agent thereby decreasing the therapeutic dose range and side effects. Methods: ES cells, CHLA10 and TC205 were cultured in the presence of vehicle or MIT or VIN or ETO or in combinations (MIT+VIN or MIT+ETO). After 2 days, cell viability was measured using The CellTiter-Glo® Luminescent Cell Viability Assay kit. The apoptosis induced by each of the above-mentioned treatments on the ES cells was measured by Flow cytometry using Annexin V Apoptosis Detection Kit. The expression of cleaved-Poly (ADP-ribose) polymerase (c-PARP), a marker for apoptosis was determined by Western blot analysis. Results: While all treatments showed ES cell growth inhibition, the combination treatment of MIT+ETO was more effective (significant at p Conclusion: The combination MIT+ETO caused more cell growth inhibition when compared to individual treatments in the TC205 and CHLA10 cell lines. These results demonstrate that MIT in combination with standard chemotherapeutic agents potentially increases therapeutic efficacy in ES. However, these results are limited to in vitro studies and need to be tested in an animal model to determine reproducibility and assess the toxicity.Item Combination of Vincristine and Tolfenamic Acid induces Anti-proliferative activity in Medulloblastoma Cells.(2017-03-14) Sankpal, Umesh; Hafeez, Areeba; Bowman, W. Paul; Basha, Riyaz; Patil, ShrutiBackground: Medulloblastoma (MB) is the most common pediatric malignant brain tumor and usually originates in the cerebellum. These tumors have the propensity to disseminate throughout the central nervous system and are often difficult to treat. Chemotherapy is widely accepted as part of the multimodality treatment approach for MB. However, it is associated with debilitating toxicity and potential long term disabilities. Vincristine, a commonly used chemotherapeutic agent for MB treatment, is known to induce some toxic effects including peripheral neuropathy. Reducing the dose of the drug to minimize the toxic effect also reduces the cytotoxic efficacy of the drug. Purpose: The aim of this study was to test a combination treatment involving vincristine and an anti-cancer non-steroidal anti-inflammatory drug, Tolfenamic acid (TA) against MB cell lines. Previously, we showed that TA inhibited MB cell proliferation and tumor growth in mice by targeting the transcription factor specificity protein-1 (Sp1) and an inhibitor of apoptosis protein, survivin. The overexpression of survivin is associated with aggressiveness and poor prognosis in several cancers. Methods: DAOY and D283 cells were treated with vehicle (DMSO) or low dose of vincristine (DAOY: 2ng/ml; D283: 1ng/ml) or TA (10 µg/ml) or combination of vincristine + TA and the cell viability was measured at one and two days post-treatment using Cell-TiterGlo kit. Flow cytometry was employed to analyze apoptotic cells using Annexin-V staining and cell cycle phase distribution using propidium iodide staining. The activation of apoptotic pathways was further investigated by assessing the levels of effector caspases with Caspase 3/7-Glo kit and the expression of apoptotic markers [c-PARP, Bcl2, and survivin] by Western blot (WB) analysis. The expression of key proteins associated with cell cycle [Cyclin A, B, D CDK4/6 and p21] was also determined by WB analysis. Results: When compared to individual agents, the combination of TA and vincristine increased MB cell growth inhibition which is accompanied by an induction of apoptotic markers and the modulation of proteins associated with cell cycle phase distribution. Conclusions: These results suggest that vincristine and TA combination treatment is effective for inducing anti-proliferative response in MB cells. The experiments to evaluate the effect of this combination in animal model for MB are currently under study.Item Copper Tolfenamic acid induces anti-proliferative activity effective against Medulloblastoma cells(2018-03-14) Sankpal, Umesh; Bowman, W. Paul; Basha, Riyaz; Arechiga, BiancaPurpose: Medulloblastoma (MB) is the most common pediatric malignant brain tumor, comprising 20% of all childhood brain tumors. Between 250-500 children per year are diagnosed in the US alone. Standard therapies result in severe long-term morbidities. Therefore, there is an urgent need for inventing novel effective treatment strategies with lower side-effects. Our laboratory showed anti-cancer activity of Tolfenamic acid (TA) in pre-clinical model for MB. Recent studies showed higher pharmacological effect of TA when synthesized as a complex with copper (Copper-TA, Cu-TA). Our aim was to investigate the anti-cancer activity of Cu-TA against MB cell lines. We hypothesize that Cu-TA presents higher anti-cancer activity and is more effective than TA to induce cytotoxicity against MB cells. Methods: DAOY and D283 cells were obtained from ATCC and grown following standard cell culture conditions. Cells were treated with TA or Cu-TA and the cell viability was measured at 24 and 48 h post-treatment using a CellTiter-Glo kit. The induction of apoptosis was investigated by studying caspase activation using the Caspase 3/7-Glo kit. In addition, reactive oxygen species (ROS) involvement was measured by flow cytometry. Results: Both Cu-TA and TA treatment resulted in decreased cell viability. However, when compared to TA, Cu-TA was more effective at inducing anti-proliferative activity in MB cells. Cu-TA induces increased production of ROS. The anti-proliferative activity of Cu-TA was accompanied by an increase in Caspase 3/7 activity, suggesting the induction of apoptosis. Conclusions: Cu-TA was more effective than TA. Therefore, it has potential as an effective anti-cancer agent for inhibiting MB cell growth. Further studies are needed to better understand Cu-TA’s mechanism of action.Item Determining the cytotoxicity of Clotam and Copper-Clotam against Cardiomyocytes(2021) Siraj, Sohail; Patel, Krishna; Mukka, Lasya; Sankpal, Umesh; Basha, RiyazBackground: Chemotherapy (ChT) is required in the treatment of many cancers. Most ChT agents exhibit unwanted side-effects by causing damage to healthy cells. Side effects from many common ChT agents are leaving pediatric cancer survivors with lasting organ system damage, specifically damage to the heart. Past studies conducted by our group demonstrated the anti-cancer activity of clotam (tolfenamic acid-TA) and copper-clotam (CuTA). Our laboratory demonstrated the anti-cancer activity of CuTA against several cancer cell lines. CuTa is showing higher cytotoxicity against cancer cells even at much lower dose than TA. Our long term objective is to test CuTA to sensitize cancer cells to ChT. Methods: Cardiomyocytes H9c2 (cell line derived from rat heart tissue) originally obtained from the American Type Culture Collection (Manassas, VA) were cultured as per the supplier's instructions. H9c2 cells were treated with TA or Cu-TA or Doxorubicin and combinations (for example, TA and Doxorubicin) and cell viability assay was measured using CellTiterGlo (Promega) kit at 48 hours post-treatment following manufacturer's instructions. Results & Conclusion: We found that TA or CuTA were not cytotoxic in H9c2 cells at tested doses. TA kept more cells alive in conjunction with Doxorubicin than did the control. Our results also demonstrate that the IC50 values of TA and CuTA, determined with cancer cell lines, are not toxic to H9c2 cells. These results provide evidence that CuTA does not induce toxicity in cardiomyocytes and supports further testing for translational application in combination therapy with ChT.Item Evaluating new treatment strategy for medulloblastoma(2021) Mohammad, Shanzay; Basha, Raasil; Sankpal, UmeshPurpose: Medulloblastoma is the most common malignant pediatric brain tumor. Although chemotherapy, radiation, and surgical resection are often successful, patients in remission often suffer from long-term side effects and face the potential for re-growth of their cancer. This raises a need for effective, less toxic therapeutic strategies. An anti-apoptotic protein, Survivin, and its transcription factor, Sp1, are over-expressed in most cancers including medulloblastoma and are associated with a poor prognosis. Metformin, an anti-diabetic drug with relatively low toxicity profile, was found to have anti-cancer properties in many cancers. This study was conducted to evaluate the effects that Metformin has on the inhibition of medulloblastoma cells. We hypothesize that Metformin treatment will decrease the growth of medulloblastoma cells, possibly by inhibiting Survivin expression through Sp1 transcription factor downregulation. Methods: Human medulloblastoma cells were treated with increasing Metformin doses, then cell viability was assessed at 24 and 48 hours post-treatment using a CellTiter-Glo cell viability assay. Survivin and Sp1 expression in Metformin-treated cells was assessed by Western blot analysis. Results: As the concentration of Metformin increased, the number of metabolically active cells decreased. Sp1 and Survivin protein levels were reduced by Metformin in a dose/time dependent manner. Conclusion: The results validated that Metformin treatment resulted in reduced cell proliferation and decreased Survivin expression. Given that high Survivin levels are associated with resistance to chemotherapy and radiation, future studies will evaluate whether inhibiting Survivin using Metformin will sensitize cancer cells to chemotherapy.Item Evaluating the anti-leukemic effect of clotam and copper-clotam using CCRF-CEM cell lines(2021) Patel, Krishna; Siraj, Sohail; Basha, Riyaz; Sankpal, UmeshPurpose: Acute lymphoblastic leukemia (ALL) is the most common type of cancer in children younger than 5 years. Patients with ALL have bone marrow that produces immature white blood cells, which are unable to effectively fight infections. NSAIDs are common pain reliving agents that act through COX inhibition, which stops the production of prostaglandins. Clotam (Tolfenamic Acid/TA) is an NSAID that has anti-tumor proliferative effects. It works through targeting specificity protein (Sp) transcription factors that assist cancer cells in inhibiting apoptosis. Our objective is to test TA and copper-TA (Cu-TA), a derivative of TA, to induce an anti-leukemic response. Methods: The T-cell ALL cell line CCRF-CEM was obtained from the American Type Culture Collection (Manassas, VA) and cells were cultured as per the supplier's instructions. A cell viability assay was performed in which cells were plated in a 96-well plate and treated with increasing concentrations of TA and Cu-TA. After 48-hours, the cells were lysed, and the amount of ATP in the cells was measured using luminescence. Using this data, IC50 values were calculated. Results: The IC50 values showed both TA and Cu-TA had anti-cancer proliferative effects. Cu-TA was 15 times more potent than TA in its ability to kill CCRF-CEM cells. Conclusion: Our results demonstrate that Cu-TA is more effective than TA for killing CCRF-CEM cells. This study suggests better implications of Cu-TA in ALL therapy, if further tested using pre-clinical models.Item Evaluation of Metformin as an anti-cancer agent in Medulloblastoma(2018-03-14) Basha, Riyaz; Bowman, W. Paul; Sankpal, Umesh; Payne, KristenEvaluation of Metformin as an anti-cancer agent in Medulloblastoma Purpose: Medulloblastoma (MB) is the most common malignant brain tumor in children under 16 years of age. Standard treatment, including surgery, chemotherapy, and radiation, is successful for most; however, survivors often suffer from long-term neurocognitive and growth potential related sequelae. Therefore, there is a need to understand the molecular processes regulating MB growth to find less toxic therapies. Survivin is a protein in the Inhibitor of Apoptosis Protein (IAP) family that inhibits caspase activity. Survivin is highly expressed in MB and associated with a poor prognosis. Specificity protein 1 (Sp1) is a transcription factor regulating survivin expression and is overexpressed in many cancers. Interestingly, the use of Metformin (MET), an anti-diabetic drug, correlated with decreased occurrence of several cancers. Previous studies have demonstrated its anti-cancer activity in breast cancer cells as well. The objective of this study is to test the effect of MET on MB cells in vitro. Hypothesis: We hypothesize that MET treatment decreases the growth of MB cells in a dose and time-dependent manner, possibly inhibiting the expression of survivin via downregulating Sp1. Methods: DAOY (MB cell line from American Type Culture Collection) cells were treated with increasing concentrations of MET (0, 1, 5, 10, and 20 mM). Cell viability was assessed at 24 and 48 hours post-treatment using the CellTiter-Glo cell viability assay. Survivin and Sp1 expression in MET treated cells was determined by Western blot analysis. Potential mechanism of cell proliferation inhibition was investigated by measuring the induction of reactive oxygen species (ROS) through Flowcytometry. Results: MET treatment resulted in decreased cell viability in a dose and time dependent manner. MET treatment also decreased Sp1 and survivin expression indicating that the effect of MET is mediated via Sp1 transcription factor. We also observed MET induced cellular ROS formation, which could be a potential anti-cancer mechanism. Conclusion: Our data demonstrates that MET can inhibit MB cell growth, possibly via targeting Sp1 to down-regulate survivin and inducing ROS. We conclude that MET has the potential to be used in the treatment of MB. Due to limitations of using Metformin alone as an anti-cancer agent, additional experiments are underway to determine its use in conjunction with MB specific chemotherapeutic agents.Item Evaluation of Stability and Anti-cancer activity of Copper(II) Tolfenamic Acid with an emphasis on Pancreatic(2018-03-14) Sankpal, Umesh; Patel, Rafid; Chhabra, Jaya; Brown, Deondra; Gurung, Raj; Holder, Alvin; Rajasekhar, Maram; Basha, Riyaz; Hurtado, MyrnaPurpose: Tolfenamic acid (TA) acts as an anti-cancer agent in several cancer models via down-regulating transcription factors Sp1 and Sp3, and an inhibitor of apoptotic protein, survivin. Copper (Cu) is an important element with multiple biological functions and has gained interest in medical applications. Recently, Cu-TA has been synthesized and tested for enhanced therapeutic activity. In this study, Cu-TA was investigated for its stability and anti-cancer activity using several cancer cell lines and mouse model for pancreatic cancer (PC). Method: Cu-TA was synthesized and characterized by UV visible spectroscopy and Fourier-transform infrared spectroscopy (FTIR). Anti-proliferative activity was evaluated against twelve cell lines representing six (breast, colon, glioblastoma, medulloblastoma, pancreatic and prostate) cancers using the CellTiter-Glo kit and compared with TA. Further studies were performed using PC cells. The expression of Sp1, Sp3 and survivin was determined by Western blot and qPCR. The stability of Cu-TA was determined using 8-12 month-old powder and six-month-old stock solution. Cardiomyocytes (H9C2) were used to test the cytotoxicity in non-malignant cells. Athymic mice were injected with PC cells and treated with vehicle (control) or 25 or 50 mg/kg of Cu-TA 3 times/week and the effect on tumor growth was monitored for 4 weeks Animals body weight changes were also observed to determine overt toxicity. Results: Cu-TA significantly more effective than TA against all tested cancer cells. The IC50 values of Cu-TA were 30 to 80% less when compared with TA. Comparison of the twelve-month-old powder and six-month-old stock solution using the Panc1 cells showed similar IC50values ( Conclusion: These in vitro and in vivo studies demonstrate that Cu-TA is more effective than TA and potentially useful as an effective anti-cancer agent.Item Exosomal Proteins as Potential Markers for Breast Cancer Disparity(2022) Kandukuri, Prathima; Sankpal, UmeshPURPOSE: Breast cancer is the most common non-cutaneous malignancy and the second most lethal form of cancer among women in the United States. However, the mortality differs among different races, with Black women having significantly higher mortality rates than White women, even when factors like socioeconomic status are controlled. One explanation for this is the fact that Black women are diagnosed at higher rates with a particularly aggressive subtype known as triple-negative breast cancer (TNBC), which lacks the receptors that chemotherapy drugs classically target. It is important to recognize the various biological factors that contribute towards this health disparity. The overarching goal of this research is to identify differentially expressed exosomal proteins as potential makers to understand this disparity. Proteins under investigation include anti-apoptotic proteins and transcription factors. The specific goal for this project is to optimize protocol for exosome isolation and characterization. METHODS: MDA-MB-231 and MDA-MB-468 metastatic TNBC cell lines were cultured in media supplemented with exosome-depleted serum. After 24h, the culture media was centrifuged to remove cell debris and processed for exosome isolation using ExoQuick-TC kit (System Biosciences). The protocol involves precipitation of exosomes using the ExoQuick reagent. The exosomal pellet was resuspended in PBS and used for Western blot to analyze proteins and Nanoparticle Tracking Analysis (NTA) for determining exosomes size and concentration. The NTA calculates the size of the particles based on their movement. For Western blotting the antibodies used were against standard exosomal markers. RESULTS: Western blot analysis for exosomes showed that all samples were positive for standard exosomal protein markers such as Flotillin, CD54 and EpCAM and negative for non-exosomal marker GM130. The average size of the exosomes isolated from the MDA-MB-231 and MDA-MB-468 were 147.8 nm, and 146.6 nm respectively as determined by NTA. The concentration of exosomes from the two cells lines was determined to be 3.01e+8 ± 7.96e+6 particles/mL for MDA231 and 1.61e+8 ± 8.35e+6 particles/mL for MDA468. CONCLUSIONS: This research project streamlined a method to isolate and characterize exosomes from TNBC cell lines using the ExoQuick kit. The next step would be to isolate exosomes from a panel of breast cancer cell-lines as well from serum or plasma derived from White and Black breast cancer patients. Exosomes from these racial groups will be probed to investigate the differential expression of any potential biomarker. Such markers can be developed into novel diagnostic tools or as potential therapeutic targets, which will help clarify and reduce the current mortality gap between the two populations.Item EXPLORING A COMBINATION USING CHEMOTHERAPY AND TOLFENAMIC ACID TO INDUCE ANTI-PROLIFERATIVE RESPONSE IN MEDULLOBLASTOMA CELL LINES(2024-03-21) Chen, Liling; Sankpal, Umesh; Basha, RiyazBackground: Medulloblastoma (MB) is the most common malignant brain tumor in children, with a peak incidence between the ages of 5-9 years old. Originating in the cerebellum, it often metastasizes throughout the CNS via the CSF, making it very difficult to treat. Current treatment options are limited, and includes surgical resection followed by radiation and chemotherapeutic agents. However, these agents are associated with numerous toxicities and long-term neurocognitive deficits in survivors. Our laboratory is interested in identifying drug resistance cell markers and combination therapies that target them to help increase efficacy of the chemotherapeutic agents. We have previously demonstrated that combination therapy of chemotherapeutic agents with Tolfenamic acid (TA), decreased the number of viable cancer cells when compared to the chemotherapeutic agents alone. TA is a non-steroidal anti-inflammatory drug, and its anti-cancer activity can be attributed to its ability to downregulate Specificity Protein 1 (Sp1), a transcription factor responsible for the upregulation of the anti-apoptotic protein, Survivin. Numerous cancerous tumors have been known to express high levels of Sp1 and SurvivIn, however these markers have not been well established in MB. Purpose: The aim of the project is to elucidate the association of Survivin expression in MB cells with the likelihood of patient survival and to test combination treatments of chemotherapeutic agents with the potential Survivin inhibitor, TA. The goal is to reduce the effective dosage of the chemotherapeutic agent Cisplatin, thereby reducing their side effect profiles. Methods: A R2 genomics visualization platform was accessed to obtain data regarding patient survival rates and Survivin expression in MB cells. A Kaplan-Meier curve was then generated to analyze the relationship. MB cell lines, DAOY, and D283 cells were obtained through the ATCC and cultured following standard cell culture conditions. Cells were then treated with vehicle (DMSO) or optimized doses of a chemotherapeutic agents (Vincristine or Cisplatin) with TA. Cell viability was measured at 24h and 48h post-treatment using Cell-TiterGlo kit (Promega) following manufacturer’s instructions. Results: The Kaplan-Meier curve showed that the overexpression of Survivin resulted in a poor prognosis and low survival rates among MB patients. Compared to results of MB cell inhibition with individual agents (Vincristine and Cisplatin) from our previous studies, the combination of TA and Vincristine or TA and Cisplatin showed decreased MB cell growth and downregulation of Survivin. Differential effects of Vincristine and Cisplatin were noted against DAOY and D283 cell lines. Conclusion: These preliminary observations suggest that Survivin expression may be associated with poor prognosis in MB patients and that inhibiting Survivin with TA is inducing the anti-proliferative effects of chemotherapeutic agents in MB cells. Further research involving other chemotherapies is required to understand TA’s role in Survivin inhibition. Understanding the specific effect of Cisplatin can help to design the therapies based on the molecular sub-group of MB.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 In Vitro Methods for Evaluating Anti-Cancer Drugs(2020-12) Bowns, Jackson T.; Sankpal, Umesh; Simecka, Jerry W.; Gwirtz, Patricia A.Cancer is a major health concern for the world, with children's cancers being particularly devastating. Children's brain cancers are treatable, but those treatments often leave behind developmental impairments and so it is vital to seek out novel cancer treatment options. Examining old drugs for cancer treatment saves considerable cost and time in drug development, and so it is an important option to explore. The focus of this practicum was learning in vitro methods of evaluating potential anti-cancer drugs, using cell viability, Western Blotting, and apoptosis analyses. These methods are useful for quickly measuring changes in cancer cell death, exploring reasons for those changes via protein analysis, and can lead to further investigations. This thesis discusses current problems with medulloblastoma treatment and investigates the potential use of metformin in medulloblastoma treatment. The results of this practicum conclude that metformin is likely to be an inhibitor of medulloblastoma viability and should be further investigated.Item In Vitro Study to Identify a Novel Combination Treatment for Ewing Sarcoma(2016-03-23) Sankpal, Umesh; Bowman, Paul; Ray, Anish; Conjeevaram Nagarajan, Bhavani Saranya; Basha, Riyaz; Gotlib, DanielIntroduction: Ewing sarcoma (ES) is an aggressive tumor that predominantly occurs in young adolescent populations. Chemotherapy used to treat ES is associated with long-term morbidity. The objective of this study was to identify an effective combination treatment option for treating ES. A fusion protein EWS-FLI1 is associated with [greater than] 85% of ES incidences and mithramycin (Mit) is known to target this fusion protein and is currently in clinical trials. We investigated the combination of Mit to induce the efficacy of Doxorubicin (Dox), a widely used chemotherapeutic agent for treating ES patients. Methods: ES cell lines CHLA9, CHLA10, and TC71 were cultured in laboratory and treated with increasing concentration of Mit or Dox. Cell viability was assessed using CellTiter-Glo luminescent assay at 48 hour post-treatment. Optimized doses for Mit and Dox were used for combination treatment. Western blot analysis was used to evaluate the apoptotic markers, caspase 3, cleaved PARP and Bcl2. Real-Time PCR was used to evaluate the expression of EWS-FL1 downstream targets. RNA was extracted using Trizol reagent (Invitrogen) and subjected to cDNA synthesis using Superscript III reverse transcriptase. PCR was carried out with cDNA using TaqMan primer-probes specific for ID2, LDB2, NROB1 and NCOR1. Results: The results of current study illustrated that Mit or Dox treatment resulted in a dose and time dependent decrease in ES cell proliferation. Mit down-regulated the EWS-FLI1 downstream targets (ID2, LDB2, NROB1 and RCOR1) in TC71 cells. Combination of Mit+Dox enhancesd the anti-proliferative effect in ES cell lines which was accompanied by modulation of apoptotic markers. Conclusion: This investigation provides evidence for the use of Mithramycin for enhancing the efficacy of chemotherapeutic agents. In vivo assays are underway to confirm in vitro results. These pre-clinical studies will have clinical implications for treating Ewing Sarcoma patients. The proposed combination to induce chemotherapy response is highly helpful since it can be tested to reducing chemotherapy dose(s) and addressing the issues related to side-effects.