More than meets the eye: exploring Sonic Hedgehog medulloblastoma from a neurodevelopmental perspective

Cerebellar development is a complex process that requires tight orchestration of various factors to ensure proper cerebellar function. The relatively long time-span for the cerebellum to reach maturity makes the cerebellum vulnerable to developmental dysregulation, which can ultimately manifest in the malignant transformation into medulloblastoma, one of the most frequently diagnosed childhood brain tumors. This thesis aimed at addressing some of the complex interplay of the molecular players during cerebellar development and how they may facilitate tumor formation.In Chapter 1, the development of cerebellar granule cells, the most abundant cell type of the brain, is described based on current literaturereviewed in Chapter 1. The predecessorsir progenitors of these granule cells (e.g., cerebellar granule neuron progenitors or CGNPs) are known to be the cell-of-origin for at least two subtypes of medulloblastoma. In Chapter 2, we studied the dynamic changes in gene expression in murineouse CGNPs during brain development and shed light on the age-specific onset and pathway mutations that are associated with SHH medulloblastoma. In Chapter 3, we explored the role of transcription factor CREB in CGNP development and medulloblastoma therapy, since we had discovered that CREB phosphorylation activity status strongly correlates with patient survival in SHH and Group 3 medulloblastoma. In Chapter 4, we assessed the role contribution genes encoding histone modifier complexes, theof histone modifier KMT2D into medulloblastoma pathogenesis. In the last experimental chapter (Chapter 5), we set out to establish primary pediatric brain tumor cell cultures that faithfully maintain tumor characteristics, which can be employed as models for understanding tumor biology and therapeutic discovery. Finally, we summarized our findings and discussed their implications in Chapter 6

Effects of Peel Extract from Citrus reticulata and Hesperidin, A Citrus Flavonoid, on Macrophage Cell Line

The extract of Citrus reticulata has been studied for its biological activities, due to its citrus flavonoid content. The extract and its flavonoid compounds exhibit growth inhibition properties in several cancer cell lines and in vivo models. Conversely, the extract can also induce cell proliferation and angiogenesis, and shows estrogenic effects, in vitro and in vivo. Because of the contrasting effects that depend on the concentration or dosage, the precise action of the extract and its flavonoids need to be elucidated in various cell types. The objective of this study is to evaluate the effect of Citrus reticulata peel extract (Citrus extract) and hesperidin, a citrus flavonoid, on the modulation of cell proliferation in the RAW 264.7 macrophage cell line. Cell viability under Citrus extract or hesperidin treatment was assessed by using the MTT assay. The expression of interleukin-10 (IL-10), an anti-inflammatory cytokine, modulated by Citrus extract was also examined by immunostaining. Low concentrations of Citrus extract at 1 and 100 μg/mL were able to induce cell proliferation, though not significantly, as shown by cell viability of 138 and 114%, respectively. At higher concentrations of 500, 750, and 1000 μg/mL, Citrus extract decreased cell viability significantly by up to 64, 46, and 36%, respectively. Accordingly, hesperidin at low (3.1 μg/mL−61.1 μg/mL) or high (152.6 μg/mL−305.3 μg/mL) concentrations increased or reduced cell viability significantly by up to 116−136% or 10−61%, respectively. The value of the 50% inhibitory concentration (IC50) of Citrus extract was more than three times higher (756 μg/mL) than that of hesperidin (203 μg/mL = 332 μM). Additionally, 250 μg/mL of Citrus extract was able to induce IL-10 expression compared with the control. These results demonstrate that Citrus extract and hesperidin exert a biphasic effect on macrophage cells. The future development of Citrus extract as a co-chemotherapeutic, anticancer, or immunomodulatory agent should include careful consideration of its biphasic effect on each cell type

Synergistic Combination of Ciplukan (Physalis angulata) Herbs Ethanolic Extract and Doxorubicin on T47D Breast Cancer Cells

Doxorubicin is one of chemotherapeutic agent widely used in breast cancer treatment, but in high dose doxorubicin gives negative side effect, including vomit, nausea, immune suppression, and cardiac toxicity. This toxicity hopefully could be reduced by combination chemotherapy using natural herbs such as ciplukan herb. This research was conducted to explore cytotoxic activity of single ciplukan herbs ethanolic extract and its combination with doxorubicin on T47D breast cancer cells. Cytotoxic activity of ciplukan herbs ethanolic extract only and its combination with doxorubicin were tested on T47D cells using MTT assay to obtain IC50 value and combination index (CI), respectively. Single extract showed cytotoxic activity on T47D cells with IC50 value of was 160 µg/ml. Thus, combination treatment from ciplukan herbs ethanolic extract and doxorubicin showed synergistic effect (CI<1,0). This effect was reached at concentration of ciplukan herbs ethanolic extract-doxorubicin 80 μg/ml- 2 nM, 80 μg/ml-4 nM, and 80 μg/ml-8 nM. This research indicated that ciplukan herbs ethanolic extract is potential to be applied as co-chemotherapeutic agent in breast cancer therapy.Key word : ciplukan herbs, doxorubicin, co-chemotherapy, T47D cell

POTENSI EKSTRAK ETANOLIK KULIT BUAH JERUK NIPIS (Citrus aurantiifolia (Cristm.) Swingle) SEBAGAI AGEN KHEMOPREVENTIF MELALUI PENEKANAN EKSPRESI c-Myc DAN PENGHAMBATAN PROLIFERASI PADA SEL PAYUDARA TIKUS GALUR SPRAGUE DAWLEY TERINDUKSI 7,12-DIMETILBENZ[a]ANTRASENA

The using of natural-based medicine is growing rapidly in societies. Besides being cheap and affordable, natural-based medicine is relatively safer than the synthetic drugs. Peel of Citrus aurantifolia (Cristm.) Swingle) is one of the chemopreventive agent which contain flavonoids have potency as anticarcinogenic agent. This study is designed to study the potency of Citrus aurantifolia peel ethanolic extract in proliferation inhibition of Rattus norvegicus mammary cell of Sprague Dawley strain which is induced by 7,12-Dimethylbenz[a]anthracene (DMBA). Rats were divided into five groups consist of DMBA treatment, CMC-Na treatment, extract 1500 mg/kgBW treatment, treatment of DMBA+ extract 750 mg/kgBW and DMBA+ extract 1500 mg/kgBW. At the beginning of the tenth week of the study, breasts was isolated and stored in 10% formalin buffer. Observation of cell proliferation was done by AgNOR method. C-Myc expression observed using immunohistochemistry (IHC). Observation of mammary cell with AgNOR method indicated that the treatment of Citrus aurantifolia peel ethanolic extract can inhibit cell proliferation significantly. Dosage 1500 mg/kgBW gave higher inhibition effect than dosage 750 mg/kgBW. IHC result showed that treatment of Citrus aurantifolia peel ethanolic extract decrease the expression of c-Myc. Dosage 750 mg/kgBW gave lower decreasing effect than dosage 1500 mg/kgBW. Citrus aurantifolia peel ethanolic extract inhibited the proliferation of mammary cell induced DMBA through the inhibition of c-Myc expression in dose dependent phenomena so that it is a potential chemopreventive agent.POTENSI  EKSTRAK ETANOLIK KULIT BUAH JERUK NIPIS (Citrus aurantiifolia (Cristm.) Swingle) SEBAGAI AGEN KHEMOPREVENTIF MELALUI PENEKANAN EKSPRESI c-Myc DAN PENGHAMBATAN PROLIFERASI PADA SEL PAYUDARA TIKUS GALUR SPRAGUE DAWLEY TERINDUKSI 7,12-DIMETILBENZ[a]ANTRASENA POTENCY OF CITRUS PEELS (Citrus aurantiifolia (Cristm.) Swingle) ETHANOLIC EXTRACT AS CHEMOPREVENTIVE  AGENT THROUGH DOWNREGULATION OF           c-Myc EXPRESSION AND INHIBITION OF 7.12-DIMETHYLBENZ[a]ANTRACHENE INDUCED FEMALE  SPRAGUE DAWLEY RATS BREAST CELL PROLIFERATION Dewi Pratiwi, Novi Hastuti, Niken Nur W, Inna Armandari, Muthi’ Ikawati,Adam Hermawan  dan Edy Meiyanto*)Cancer Chemoprevention Research Center Fakultas Farmasi, Universitas Gadjah Mada ABSTRAK Penggunaan obat berbasis alam saat ini berkembang pesat di semua kalangan masyarakat. Selain karena harga yang lebih terjangkau, obat berbasis alam relatif lebih aman dibandingkan dengan obat sintetik. Kulit jeruk nipis (Citrus aurantiifolia) merupakan salah satu obat berbasis alam mengandung flavonoid yang berpotensi sebagai antikarsinogenesis.  Penelitian ini dirancang untuk mengkaji potensi kulit jeruk nipis (C. aurantiifolia) dalam menekan proliferasi sel  payudara tikus galur Sprague Dawley yang terinduksi 7,12-Dimetilbenz[a]Antrasena (DMBA).  Dalam penelitian ini, tikus dibagi menjadi lima kelompok yakni kelompok perlakuan DMBA, kelompok perlakuan CMC-Na, kelompok perlakuan ekstrak dosis 1500 mg/kgBB , kelompok perlakuan DMBA+ekstrak dosis 750 mg/kgBB dan perlakuan DMBA+ekstrak dosis 1500 mg/kgBB. Pengamatan proliferasi sel payudara dengan metode AgNOR menunjukkan bahwa pemberian ekstrak kulit C. aurantiifolia dapat menekan proliferasi sel secara signifikan. Secara kuantitatif signifikansi yang dihasilkan dosis  1500 mg/kgBB lebih tinggi daripada dosis 750 mg/kgBB. Hasil pengamatan imunohistokimia pada ekspresi c-Myc mendukung data sebelumnya. Pada kelompok dosis 750 terlihat warna coklat pada sitosol yang lebih intens dibanding kelompok dosis 1500. Ekstrak etanolik kulit jeruk nipis dapat menekan proliferasi sel payudara terinduksi DMBA, penekanan proliferasi tersebut meningkat seiring peningkatan dosis sehingga jeruk nipis dapat digunakan sebagai agen khemopreventif

Potency of Citrus Peels (Citrus Aurantiifolia (Cristm.) Swingle) Ethanolic Extract as Chemopreventive Agent Through Downregulation of C-myc Expression and Inhibition of 7.12-dimethylbenz[a]antrachene Induced Female Sprague Dawley Rats Breast Cell Proliferation

The using of natural-based medicine is growing rapidly in societies. Besides being cheap and affordable, natural-based medicine is relatively safer than the synthetic drugs. Peel of Citrus aurantifolia (Cristm.) Swingle) is one of the chemopreventive agent which contain flavonoids have potency as anticarcinogenic agent. This study is designed to study the potency of Citrus aurantifolia peel ethanolic extract in proliferation inhibition of Rattus norvegicus mammary cell of Sprague Dawley strain which is induced by 7,12-Dimethylbenz[a]anthracene (DMBA). Rats were divided into five groups consist of DMBA treatment, CMC-Na treatment, extract 1500 mg/kgBW treatment, treatment of DMBA+ extract 750 mg/kgBW and DMBA+ extract 1500 mg/kgBW. At the beginning of the tenth week of the study, breasts was isolated and stored in 10% formalin buffer. Observation of cell proliferation was done by AgNOR method. C-Myc expression observed using immunohistochemistry (IHC). Observation of mammary cell with AgNOR method indicated that the treatment of Citrus aurantifolia peel ethanolic extract can inhibit cell proliferation significantly. Dosage 1500 mg/kgBW gave higher inhibition effect than dosage 750 mg/kgBW. IHC result showed that treatment of Citrus aurantifolia peel ethanolic extract decrease the expression of c-Myc. Dosage 750 mg/kgBW gave lower decreasing effect than dosage 1500 mg/kgBW. Citrus aurantifolia peel ethanolic extract inhibited the proliferation of mammary cell induced DMBA through the inhibition of c-Myc expression in dose dependent phenomena so that it is a potential chemopreventive agent

Chromosomal Instability Characterizes Pediatric Medulloblastoma but Is Not Tolerated in the Developing Cerebellum

Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHH alpha. This suggests that chromosomal instability (CIN), the process leading to aneuploidy, is an important player in medulloblastoma pathophysiology. However, it is not known if there is ongoing CIN in medulloblastoma or if CIN affects the developing cerebellum and promotes tumor formation. To investigate this, we performed karyotyping of single medulloblastoma cells and demonstrated the presence of distinct tumor cell clones harboring unique copy number alterations, which is suggestive of ongoing CIN. We also found enrichment for processes related to DNA replication, repair, and mitosis in both SHH medulloblastoma and in the highly proliferative compartment of the presumed tumor cell lineage-of-origin, the latter also being sensitive to genotoxic stress. However, when challenging these tumor cells-of-origin with genetic lesions inducing CIN using transgenic mouse modeling, we found no evidence for large chromosomal aberrations in the cerebellum or for medulloblastoma formation. We therefore conclude that without a background of specific genetic mutations, CIN is not tolerated in the developing cerebellum in vivo and, thus, by itself is not sufficient to initiate medulloblastoma

CREB signaling activity correlates with differentiation and survival in medulloblastoma

While there has been significant progress in the molecular characterization of the childhood brain cancer medulloblastoma, the tumor proteome remains less explored. However, it is important to obtain a complete understanding of medulloblastoma protein biology, since interactions between proteins represent potential new drug targets. Using previously generated phosphoprotein signaling-profiles of a large cohort of primary medulloblastoma, we discovered that phosphorylation of transcription factor CREB strongly correlates with medulloblastoma survival and associates with a differentiation phenotype. We further found that during normal cerebellar development, phosphorylated CREB was selectively expressed in differentiating cerebellar granule neuron progenitor (CGNP) cells. In line, we observed increased differentiation in CGNPs treated with Forskolin, Bmp6 and Bmp12 (Gdf7), which induce CREB phosphorylation. Lastly, we demonstrated that inducing CREB activation via PKA-mediated CREB signaling, but not Bmp/MEK/ERK mediated signalling, enhances medulloblastoma cell sensitivity to chemotherapy

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development

Intratumoral steroidogenesis in castration-resistant prostate cancer: a target for therapy

Development of castration-resistant prostate cancer (CRPC) in a low androgen environment, arising from androgen deprivation therapy (ADT), is a major problem in patients with advanced prostate cancer (PCa). Several mechanisms have been hypothesized to explain the progression of PCa to CRPC during ADT, one of them is so called persistent intratumoral steroidogenesis. The existence of intratumoral steroidogenesis was hinted based on the residual levels of intraprostatic testosterone (T) and dihydrotestosterone (DHT) after ADT. Accumulating evidence has shown that the intraprostatic androgen levels after ADT are sufficient to induce cancer progression. Several studies now have demonstrated that PCa cells are able to produce T and DHT from different androgen precursors, such as cholesterol and the adrenal androgen, dehydroepiandrosterone (DHEA). Furthermore, up-regulation of genes encoding key steroidogenic enzymes in PCa cells seems to be an indicator for active intratumoral steroidogenesis in CRPC cells. Currently, several drugs are being developed targeting those steroidogenic enzymes, some of which are now in clinical trials or are being used as standard care for CRPC patients. In the future, novel agents that target steroidogenesis may add to the arsenal of drugs for CRPC therapy