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

Shruti Patil, Umesh Tanaji Sankpal, Myrna Hurtado, Paul Bowman, Jeffrey Murray, Kathleen Borgmann, Anuja Ghorpade, Robert Sutphin, Don Eslin, Riyaz Mahammad Basha

Research output: Contribution to journalArticleResearchpeer-review

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 IC 50 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 (G 2 /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.

Original languageEnglish
Pages (from-to)67-76
Number of pages10
JournalGene
Volume705
DOIs
StatePublished - 15 Jul 2019

Fingerprint

Medulloblastoma
Vincristine
tolfenamic acid
Cell Cycle
Apoptosis
Activation Analysis
Neoplasms
Propidium
Annexin A5
Growth
Microtubules
Pharmaceutical Preparations
Cell Division
Cerebrospinal Fluid
Molecular Biology
Cell Survival
Flow Cytometry
Therapeutics
Western Blotting
Cell Proliferation

Keywords

  • Combination Index
  • Medulloblastoma
  • Survivin expression
  • Tolfenamic acid
  • Vincristine

Cite this

Patil, Shruti ; Sankpal, Umesh Tanaji ; Hurtado, Myrna ; Bowman, Paul ; Murray, Jeffrey ; Borgmann, Kathleen ; Ghorpade, Anuja ; Sutphin, Robert ; Eslin, Don ; Basha, Riyaz Mahammad. / Combination of clotam and vincristine enhances anti-proliferative effect in medulloblastoma cells. In: Gene. 2019 ; Vol. 705. pp. 67-76.
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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{\circledR} (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 IC 50 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 (G 2 /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.",
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Combination of clotam and vincristine enhances anti-proliferative effect in medulloblastoma cells. / Patil, Shruti; Sankpal, Umesh Tanaji; Hurtado, Myrna; Bowman, Paul; Murray, Jeffrey; Borgmann, Kathleen; Ghorpade, Anuja; Sutphin, Robert; Eslin, Don; Basha, Riyaz Mahammad.

In: Gene, Vol. 705, 15.07.2019, p. 67-76.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Combination of clotam and vincristine enhances anti-proliferative effect in medulloblastoma cells

AU - Patil, Shruti

AU - Sankpal, Umesh Tanaji

AU - Hurtado, Myrna

AU - Bowman, Paul

AU - Murray, Jeffrey

AU - Borgmann, Kathleen

AU - Ghorpade, Anuja

AU - Sutphin, Robert

AU - Eslin, Don

AU - Basha, Riyaz Mahammad

PY - 2019/7/15

Y1 - 2019/7/15

N2 - 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 IC 50 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 (G 2 /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.

AB - 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 IC 50 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 (G 2 /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.

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KW - Medulloblastoma

KW - Survivin expression

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