The Research For Sensitization Effect Of L-Borneol And Dihydroartemisinin On Glioma Chemotherapy

PART 1:L-Borneol could transiently open blood-brain barrier and enhance therapeutic effect of cisplatin for glioblastoma bearing mouseBackground:Glioblastoma is the most frequent primary malignant brain tumor in adults. Its histological feature is associated with the invasion of tumor cells in surrounding normal brain tissue. Standard therapy consists of surgical resection to the extent that is safely feasible, followed by radiotherapy and chemotherapy. However, the median overall survival of patients with high-grade glioblastoma, even after surgery, radiotherapy, and chemotherapy, is approximately 22 months for anaplastic astrocytoma and 16 months for glioblastoma. Up to now, there is still lack of effective systemic therapies for this disease.Cisplatin is a platinum chemotherapeutic used in a variety of malignancies. Cisplatin has been shown to reduce the activity of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase, importantly, cisplatin has shown activity against osteosarcoma, transitional cell carcinoma, squamous cell carcinoma (SCC), melanoma, mesothelioma, carcinomatosis and germinal cell tumours. However, the curative effect of cisplatin for glioblastoma is often limited because of the low permeability of cisplatin across the blood-brain barrier (BBB) and poor cisplatin penetration into the tumor tissue. The BBB is a complex network of vasculature comprising microvessel endothelial cells along with many enzymes, efflux pumps, and transporters, which work in concert to limit the access of molecules to the central nervous system (CNS). These characteristics lead to the inability of intravenously administered anti-cancer agents to reach the brain tissue. The existence of BBB does not allow the passage of 98% of small-molecule and water-insoluble drugs and 100% of large-molecule drugs. Therefore, many scientists made a lot of effort to develop various strategies to deliver anti-cancer agents through the BBB, such as temporary disruption of the BBB, chemical modification of available chemotherapeutic agents as well as utilization of endogenous transport systems were explored to improve drug delivery across the BBB, but many of these results are not satisfactory.Borneol (C10H18O), a monoterpenoid component present in the essential oils of numerous medicinal plants, such as valerian, chamomile and lavender, has been widely used in traditional Chinese medicine (TCM) as a messenger drug. Generally, borneol includes both synthetic borneol (SB) and natural borneol (NB). SB includings L-borneol and isoborneol, while NB contains L-borneol. It has been reported that isoborneol exhibited higher mucosa stimulus and hepatotoxicity than L-borneol. Ever since NB was recorded in the Chinese Pharmacopoeia (2005 and 2010 edition), it was overwhelmingly recommended in TCM clinic compared to SB. This compound is considered a GRAS (generally regarded as safe) approved by the US Food and Drug Administration (FDA) as food flavor. Previous studies have shown that borneol can accelerate the open of BBB and enhances the delivery of nanoparticles to the brain and have neuroprotective effects. However, little is known with regard to potential auxo-action through BBB between cisplatin and L-borneol. It has not been reported that if L-borneol could promote cisplatin to prolong the survival time of tumor-burdened mice. Given that cisplatin and borneol both exert positive effects on brain tumors, the strategy of combining them to enhance therapeutic effect is rational. Therefore, in the present study, we aimed to display the ability of L-borneol to improving the permeability of cisplatin and synergize with cisplatin to treat tumor-burdened mice, at last demonstrate that L-Borneol could improve the curative effect of cisplatin.Purpose of the study:1. The study of L-Borneol opening the Blood brain barrier:analyze the Evans blue in the mouse brain tissue by fluorescence and quantify the Evans blue in the mouse brain tissue.2. To investigate whether L-borneol could cause brain edema or potential acute toxicity.3. To explore the the BBB permeability of cisplatin in the presence of L-borneol in normal brain tissue, tumor tissue. Whether application of L-borneol could increase the BBB permeability of cisplatin.4. To demonstrate that cisplatin accompanied with L-borneol may suppressing tumor growth and improve tumor-burdened mice survival.Materials and methods:1. To research the promotion effects of L-Borneol on BBB opening, Evans blue was fluorescence analysised and quantitatively measured.2.7.0T MR was used to research the promotion effects of L-Borneol on BBB opening: after the mouse was treated by L-Borneol, the blood-brain barrier permeability was analysised by 7.0T MR.3. To investigate whether L-borneol could cause brain edema, mice were sacrificed after administration L-borneol 4 h later and cerebral oedema was measured with the dry-wet weight method and represented by the water content of the brain tissues.4. To investigate whether L-borneol could cause potential acute toxicity, the righting reflex was used to assess potential acute toxicity induced by oral L-borneol.5. To explore whether application of L-borneol could increase the BBB permeability of cisplatin, electrothermal atomic absorption spectrometry was used to detect platinum in the brain tissues.6. The survival time of glioma bearing mouse was used to demonstrate that the application of L-borneol could increase BBB permeability of cisplatin and improve animal survival.Results:1. Time-effect relationship of L-Borneol transiently increasing BBB permeability. After 10 min the mice were treated with L-borneol (1,200 mg/kg), levels of EB in the mouse brain tissue was 15.77 ± 0.61 μg/g, while after 20 min the levels of EB in the mouse brain tissue was 18.55 ± 0.95 μg/g, it was a peak level, after 30 min levels of EB in the mouse brain tissue was 16.64 ±1.15 μg/g. With the time go by, the levels of EB in the mouse brain tissue of 40, 50,60 min group decreased (8.25 ± 0.57,7.718 ± 1.82, and 7.302 ± 0.94 μg/g).2. Dose-effect relationship of L-Borneol increasing BBB permeability. After 20 min the mice were treated with 300,600,900, and 1,200 mg/kg L-borneol, the levels of EB in the mouse brain tissue was 13.42 ± 1.07,12.39 ± 1.05,15.25 ± 0.82, and 18.55 ± 0.95 μg/g, the BBB permeability was significantly increased compared with the blank group (P<0.05). Our team also used the 7.0T MR to confirm that L-Bornel could increase the permeability of the brain for the Gd-DTPA.3. L-Borneol has no influence on cerebral edema. Water content in normal mice was 78.02 ±0.18%; after administered with 300,600, and 1,200 mg/kg L-borneol for 4 h, respectively, there was no obvious change in water content among all these groups including the normal control group.4. Low doses of L-Borneol has no influence on animal behavior. Obvious influence on the righting reflex is observed in 900 and 1,200 mg/kg groups. However, there is no significant difference between 300,600 mg/kg groups.5. L-Borneol could promote cisplatin transport into tumor and peritumoral tissues. Oral administration of 300 mg/kg L-borneol could increase cisplatin concentration by 0.37 ±0.12 μg/g in the peritumoral tissue and a significant increase by 0.44 ±0.13 μg/g in the tumor loci.6. L-Borneol combined with cisplatin could improve survival time of tumor-bearing mice. we find that median survival of tumor-bearing mice is significantly improved in cisplatin plus L-borneol group compared with cisplatin alone (24 ± 4.9 v.s.19.3 ± 3.9 d, P<0.05).Conclution:1. L-Bomel could induce a transient significant increase in the permeability of the brain. After 10 min the mice are treated with L-borneol, the permeability of the BBB begins to increase,20 min later the permeability of the BBB reaches the peak,30 min later the opening of the BBB decreases gradually. L-Borneol increases BBB permeability with a dose-dependance.2.Therapeutic dose of L-Borneol has no influence on cerebral edema and animal behavior.3. L-Borneol could promote cisplatin transport to the brain tissues and tumor tissues of laboratory mice and improve the survival time of tumor-bearing mice.PART 2:The chemotherapy sensitizing effect of Dihydroartemisin in inducing glioma stem cells apoptosisBackground:Glioma is the most prevalent primary malignant brain tumor in adults. Although the incidence of brain tumors is relatively low compared with other cancers, the median survival with optimal therapy is only 15 months from diagnosis, and most tumors recur within 9 months of initial treatment. The primary histological feature of malignant gliomas is invasion of the tumor cells into the surrounding normal brain tissue. To date, surgery is still the most effective treatment with curative potential, but even after standard surgery, radiotherapy, and chemotherapy, the prognosis of patients with malignant gliomas is still poor. Therefore, numerous approaches have been attempted in the search for other options, including efficient chemotherapeutic agents. Because apoptotic resistance is a major challenge that hampers the efficacy of anti-cancer treatment, there have been increased efforts in the development of innovative compounds that more effectively kill therapy-resistant tumor cells.Recent reports have characterized a group of cells, termed cancer stem cells (CSCs), within tumors that drive tumor formation and growth. Glioma stem cells have been proven to play key roles in growth, invasion, angiogenesis and immune evasion of glioma. They have also been identified as the sources of chemo-and radio-resistance. Even when the majority tumor cells have been eliminated, the GSCs survive and recreate new tumor cells. Therefore, effective therapies against gliomas, especially malignant glioblastoma (GBM), may be more benefitcial if they specifically target GSCs to achieve better clinical outcomes. Because of their resistance to chemotherapeutic agents, inhibiting the growth of GSCs has been very difficult. Recently, some natural products have been reported to kill CSCs.Dihydroartemisinin (DHA), which is an active form of the Chinese medicinal herb Artemisia annua, is a safe, effective, FDA-approved and WHO-recommended mainstay for treating malaria. Recent studies have suggested that DHA also exerts preferentially cytotoxic effects on several human malignancies, including lung cancer, ovarian cancer, hepatocellular carcinoma, and pancreatic cancer. Furthermore, GSCs are responsible for glioma recurrence, metastasis, and the high mortality of glioma patients, and GSCs are considered as an important target for developing future cancer therapies or improving the current therapeutic effects. Additionally the serine/threonine kinase Akt plays an important role in tumorigenesis. The biological significance of Akt kinase activity in GSCs has been recently established. Overexpression of p-Akt can inhibit apoptosis. Furthermore, activation of Akt can predict a poor prognosis in glioma. The phosphorylation of Akt may alter the activity of proteins, such as Caspase-3, Bcl-2 family members, nuclear factor-kappa B (NF-κB) and other transcription factors that induce or inhibit apoptosis. Based on these findings, we speculated that DHA might induce apoptosis in GSCs by inhibiting p-Akt, which is important for GSCs survival. In the present study, we investigated the effect of DHA on GSCs, with a focus on apoptosis, and examined the involvement of p-Akt and Caspase-3 in this process to elucidate the possible molecular mechanisms by which DHA acts on GSCs.Purpose of the study:1. To observe the morphology change in the DHA-treated GSCs.2. To investigate the influence of DHA on the growth of GSCs and GSC spheres formation.3. To explore the cell cycle arrest and apoptosis of GSCs after treated with DHA.4. To demonstrate that DHA could inhibit phosphorylation of Akt and activates Caspase-3 in the GSCs.Materials and methods:1. The stemness characteristics of GSCs were identified by immunofluorescence staining of stem cell markers (CD133, SOX2, and Nestin).2. The morphology of DHA-treated GSCs was observe by inverted microscope.3. The effects of DHA on cell growth was evaluated by CCK-8 assay.4. The effects of DHA on the cell cycle arrest and apoptosis of GSCs were evaluated by the cell cycle assay and Annexin-V/PI flowcytometric assay.5. To reveal the underlying mechanisms, we detected the expression levels of p-Akt and Cleaved Caspase-3 by Western blotting assay.Results:1. DHA increase the sensitivity of ACNU for glioma.2. DHA inhibits the viability of GSCs. A significant morphology change in the DHA-treated GSCs was apparent and dose-dependent. DHA exhibited a dose-dependent inhibitory effect on the GL261 cells and GSCs. Interestingly, DHA shows a greater inhibition to the GSC growth more effectively than the GL261 cell growth. DHA inhibits GSC sphere formation.3. DHA inhibits the growth of GSCs 65.7 ± 2.09% of the 80 μM DHA treated cells in G0/G1 versus 51.4 ± 2.59% of the control cells (P<0.05). In addition, incubation with 80 μM DHA 24 h induced 69.26 ± 3.29% GSCs apoptosis versus 7.9 ± 1.36% of the control group (P<0.05).4. The expression levels of p-Akt decreased significantly (P<0.05) and Cleaved Caspase-3 increased significantly in a dose-dependent manner (P<0.05) after the GSCs sphere cells were treated with 20,40 and 80μM of DHA for 24 h.Conclution:1. DHA increases the sensitivity of ACNU for GL261 glioma cells.2. DHA could influence the morphology of GSCs significantly and inhibit GSC sphere formation, resulte in Gl arrest and induce apoptosis of GSCs. DHA shows a greater inhibition to the GSC growth more effectively than the GL261 cell growth.5. DHA inhibits the activation of Akt and increases the expression of the Cleaved Caspase-3 of GSCs.