The Combination Of Menadione, Vitamin C And Satinomycin To Treat Glioma In Vitro

Gliomas are the most frequent cancer in central nervous system (CNS),malignant gliomas accounting for approximately50%of all malignant braintumors and accounting for approximately2%of all malignancies. The annualincidence of malignant glioma is approximately five new cases per100,000persons. World health organization (WHO) classification of tumors of the CNSis based on their differentiation (grade I and II low-grade and grade III and IVhigh-grade gliomas, HGG) and morphology (astrocytoma, oligodendroglioma,and mixed oligo-astrocytoma). Positive therapies include surgery, radiationtherapy and chemotherapy. Surgical resection, as the most common treatment,aims to make pathological diagnosis, reduce the number of tumor cells, improvethe symptoms and prolong the survival time of patients. Patients with recurrentHGG after surgery, radiation therapy, and chemotherapy have a poor prognosis,whose median survival is39and30weeks for grade III and grade IV glioma,respectively. HGG is easy to relapse after surgery, therefore we need to find asafer and more effective method to prevent recurrence of malignant glioma andprolong the survival time of patients.The difficulty of glioma’s treatment is how to completely cure it. Surgicaltreatment alone is always difficult to completely cure tumors, especially HGG. Itis because the diffuse infiltrative growth of tumor cell and the difficulty todistinguish malignant tumors from normal tissue in operation. Compared to normal cells with limited replication ability, cancer cells and stem cells have thepotential of unlimited replication. This feature means that the tumor cells andstem cells can be immortalized. Cancer stem cells (CSCs) may lead to tumorincidence, recurrence and drug resistance. CSC is a specific tumor subgroupwhich has self-renewal capacity and produces the heterogeneity of tumor cells.CSC may be in a dormant state for a long time and has a variety of drug-resistant molecules which can resist anti-neoplastic drug, resulting in pooroutcome of anticancer therapy. CSC can be found in a variety of human organs,for example, blood, breast, brain, bone, skin, liver, prostate, ovary, bladder,rectum, and pancreas.Salinomycin is a monocarboxylic polyether ionophore isolated fromStreptomyces albus. It is a lipophilic, anionic compound with weak acid.Salinomycin may act on different biological membrane, including cytoplasticand mitochondria membranes. As a selective ionophore, salinomycin has a goodselectivity for potassium ions and alkali ions, thereby salinomycin can improvepotassium ions outflow of the cells and mitochondria as well as inhibitmitochondrial oxidative phosphorylation. Salinomycin has good antimicrobialeffect against gram-negative bacteria. Salinomycin, as an anticancer drug, cannot only act on a variety of tumor cells but also target CSCs directly.Salinomycin in combination with other anticancer drugs has a synergistic effect,so salinpmycin is a very promising anticancer drug. In this study, we usedsalinpmycin in combination with other antineoplastic drugs to kill glioma cells in vitro, and then used senecent test to verify this combination can effectivelyinhibit glioma cell growth and regrowth.Objective: To measure the minimum effective concentration and timeneeded of the combination of menadione, vitamin C and salinomycin to kill allglioma cells without relapse in vitro. Methods:(1) Evaluate effect of thecombination of menadione, vitamin C and salinomycin in DBTRG-05MG,hGCL-9, hGCL-10and hGCL-12glioma cell lines in short-term assay by cellculture techniques. The groups of combination of menadione and vitamin Cwere divided as follows:①sterile distilled water5μl+DMSO5μl+completemedium5μl；②1μM menadione5μl+0.1mM vitamin C5μl+completemedium5μl；③2.5μM menadione5μl+0.25mM vitamin C5μl+completemedium5μl；④5μM menadione5μl+0.5mM vitamin C5μl+completemedium5μl；⑤10μM menadione5μl+1.0mM vitamin C5μl+completemedium5μl；⑥5μM menadione5μl+2.5mM vitamin C5μl+completemedium5μl. The combinations of menadione, vitamin C and salinomycin(0.5μM) are grouped as follows:①sterile distilled water5μl+DMSO5μl+0.5μM salinomycin5μl;②1μM menadion5μl+0.1mM vitamin C5μl+0.5μMsalinomycin5μl;③2.5μM menadion5μl+0.25mM vitamin C5μl+0.5μMsalinomycin5μl;④5μM menadion5μl+0.5mM vitamin C5μl+0.5μMsalinomycin5μl;⑤10μM menadion5μl+1.0mM vitamin C5μl+0.5μMsalinomycin5μl;⑥25μM menadion5μl+2.5mM vitamin C5μl+0.5μMsalinomycin5μl. The combination of menadione, vitamin C and salinomycin (1.0μM) are grouped as follows:①sterile distilled water5μl+DMSO5μl+1.0μM salinomycin5μl;②1μM menadion5μl+0.1mM vitamin C5μl+1.0μMsalinomycin5μl;③2.5μM menadion5μl+0.25mM vitamin C5μl+1.05μMsalinomycin5μl;④5μM menadion5μl+0.5mM vitamin C5μl+1.0μMsalinomycin5μl;⑤10μM menadion5μl+1.0mM vitamin C5μl+1.0μMsalinomycin5μl;⑥25μM menadion5μl+2.5mM vitamin C5μl+1.0μMsalinomycin5μl. Cells were plated in96-well microplates and reachedadherence overnight. After adding drugs at the appropriate combination andconcentration, cells were incubated for72h and cell viability was measured byCCK-8. A microplate reader with Thermo Scientific Ascent software v.2.6program was used to test the optical absorption values. Cell viability wascalculated by absorbance value of each experimental group/absorbance value ofcontrol group. Data were evaluated as mean±standard deviation. Then graphswere made to show the effect of different combinations of anticancer drugs withdifferent concentrations and time against glioma cells.(2)The minimumeffective concentration and time to kill all the glioma cells in the above cell lineswere measured in long-term assay. Drugs were added and maintained100μl perwell for2weeks (media and drugs were changed twice a week). After2weeks,cells were incubated in drug-free media (media changed twice a week) for4weeks and then observed if regrowth happened using a routine invertedmicroscope. According to the time of adding drugs, four groups were divided:3days,7days,10days and14days, respectively. Each group used three kinds of different concentrations of antineoplastic agents:①menadione2.5μM+vitaminC0.25mM+salinomycin0.5μM；②menadione5.0μM+vitamin C0.5mM+salinomycin0.5μM;③menadione7.5μM+vitamin C0.75mM+salinomycin0.5μM.(3)Cells were plated in a small petri dish and senescence-associatedβ-galactosidase staining was performed. Cell were observed under afluorescence microscope. This part was divided into four groups, a control groupand three experimental groups:①menadione2.5μM+vitamin C0.25mM+salinomycin0.5μM;②menadione5.0μM+vitamin C0.5mM+salinomycin0.5μM;③menadione7.5μM+vitamin C0.75mM+salinomycin0.5μM.Results:(1)Use the combination of menadione, vitamin C and salinomycintherapy of gliomas significant effect, the drug concentration is lower than thecombination of menadione and vitamin C. DBTRG-05MG、hGCL-9、hGCL-10and hGCL-12cell lines got similar results.(2)In4glioma cell line in thecombination of menadione、vitamin C and salinomycin at concentration of7.5μM、0.75mM、0.5μM, respectively killed all the glioma cells in7days and noregrowth in drug-free media within4weeks. However, hGCL-9and hGCL-10inthe combination of menadione、vitamin C and salinomycin at concentration of5.0μM、0.5mM、0.5μM, respectively killed all the glioma cells in10days and noregrowth in drug-free media within4weeks.(3) Observed by fluorescencemicroscopy, contral group almost no coloure change, but the experiment groupis dyed blue by senescence-associated β-galactosidase. Conclusions:(1) Thecombination of menadione，vitamin C and salinomycin can effectively treat gliomas.(2)Use regrowth concentration0(RC0) as the termination of thelong-term experimental parameters, we can get the effective concentration andtime to kill all the cancer cells. And long term assay can be observed tumorregrowth or not.(3) A new combination of menadione， vitamin C andsalinomycin can be successfully induced glioma cell senescence， so thiscombination can preventing cell proliferation and inhibittion of tumor growth.