Survivin Ser81 Plays An Important Role in PI3K/Akt/mTOR Signaling Pathway

Background: Survivin, a member of the inhibitor of apoptosis protein family, has been associated with protection from cell apoptosis and regulation of mitosis. Phosphorylated-Survivin at Ser81 was reported to provide cytoprotection against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in L929 cells by inducing a backloop activation of phosphatidylinositol 3-kinase (PI3K). Therefore Akt as a possible substrate of PI3K was investigated.Methods: L929 cells were pretreated with/without 50 μM LY294002 or 10 μM Perifosine, and infected with viral particle of Survivin, anti sense of Survivin, Ser81Ala mutated Survivin or vector only. Cells were then harvested, lysed and subjected to immunoblot assay to detect Akt, phosphorylated Akt (Ser473), mammalian target of rapamycin (mTOR), phosphorylated-mTOR (Ser2448).Results: Survivin induced Akt and mTOR phosphorylations in a viral particle concentration dependent manner. Pretreatment of LY294002 or Perifosine prior to Survivin infection, attenuated Akt or mTOR phosphorylations, respectively. Low Akt or mTOR phosphorylations were observed when L929 cells were infected with Ser81Ala mutated Survivin.Conclusion: Ser81 phosphorylation site of Survivin played an important role in activating Survivin/PKA/PI3K/Akt/mTOR signaling pathway.Keywords: survivin, Ser81, Akt, mTOR, LY294002, perifosin

A Brief Outlook on Pharmacogenetics (PGx): Focus in MicroRNAs (miRNAs) and Cancer Stem Cells (CSCs)

It has been known that there are differences in response to medications both in terms of clinical activity and side effects. Among all influencing factors, genetic variation has been considered to play a crucial part. By genetic investigation, the differences in drug metabolism, transport and target could be disclosed. Termed as “pharmacogenetics (PGx)”, that focuses on the variants within one or more candidate genes. Genetic tests have been started for screening polymorphisms prior to drug prescription, moreover many biomarkers were developed in oncology. Recent PGx investigations have been conducted to identify mRNAs, microRNAs (miRNAs) and other downstream signals that are affected by variation in genes that might cause drug response variability. Another intriguing study related to PGx in cancer stem cells (CSCs) has recently aroused. CSC shows more resistant behavior to drug. CSCs are subpopulation of cells, which share some same markers with stem cells. CSC can induce specific signal transduction pathways. Variation in genes affect CSCs activity are generally neglected in the past PGx studies. This could be one of the explanation why past PGx studies in cancer cell do not achieve optimal clinical outcome. Keywords : pharmacogenetics, pharmacogenomics, microRNAs, cancer stem cell

Human Umbilical Cord Blood Serum Has Higher Potential in Inducing Proliferation of Fibroblast than Fetal Bovine Serum

Background: Cytokines and growth factors were reported to play an important role in stimulating fibroblast proliferation. In vitro culture, fibroblast is mostly culture in medium containing fetal bovine serum (FBS).  Human umbilical cord blood (hUCB) has been reported to have low immunogenic property and potential in wound healing, so therefore hUCB serum (hUCBS) could be potential and were investigated in current study.Materials and Methods: Five hUCBs were collected from healthy volunteers with normal delivering procedure. hUCB was ex utero immediately collected from umbilical vein in vacutainers and processed. NIH3T3 cells were cultured in DMEM with 10% FBS or 5-20% hUCBS for 48 hours. Cells were then quantified using MTT assay. Protein concentration of FBS and hUCBS were quantified using Bradford assay.Results: NIH3T3 cells density grown in DMEM with 10% FBS was the lowest. NIH3T3 cells densities were increased along with the increment of hUCBS concentrations. MTT results showed that average number of NIH3T3 cells grown in DMEM with 10% FBS was 6,185±1,243. Meanwhile average numbers of NIH3T3 cells grown in DMEM with 5%, 10% and 20% hUCBS were 8,126±628, 9,685±313 and 12,200±304, respectively. Average numbers of NIH3T3 cells grown in DMEM with 5% hUCBS were significantly higher than the ones with 10% FBS (p=0.000). Bradford results showed that concentration of hUCBS was significantly higher than the one of FBS (p=0.000).Conclusion: hUCBS could induce higher proliferation rate of NIH3T3 cells than FBS. Hence hUCBS could be suggested as an alternate of FBS in inducing fibroblast.Keywords: NIH3T3, fibroblast, UCB, serum, FBS, proliferatio

Osteoclastogenesis in Periodontitis: Signaling Pathway, Synthetic and Natural Inhibitors

Osteoclast activities are responsible for the resorption of bone cells found in several bone diseases, one of which is periodontitis and arthritis. The downregulating signals of Receptor Activator of Nuclear Factor kB (RANK)-RANK Ligand and Tumor Necrosis Factor  (TNF)-a are the major cause of the bone destruction. Studies and experiments have been performed to overcome this matter. Various medications are now available to treat bone-related diseases, targeting the specific aspect of the signaling. Synthetic drugs such as denosumab and bisphosphonates have complex pharmacological action and have been the leading choice in treatment. Evidence in studies proved that natural resources including herbal products have potential application to therapy for bone loss, with caffeic acid and Caffeic Acid Phenethyl Ester (CAPE) showing significant inhibitory results and Chinese herbs such as Herba epimedii (Yín Yáng Huò) and Fructus psoraleae(Bǔ Gǔ Zhī) proved to contain components that give similar effects to estrogen. The purpose of this review is to discuss the therapy value of available synthetic and natural therapeutic agents. Understanding the mechanisms of both agents will not only clarify their function as therapeutic agents, but can also be the key to the treatment of diseases caused by bone resorption by targeting specific aspects of osteoclastogenesis.Keywords: osteoclastogenesis, TNF, RANKL, bone resorption, natural resource, signaling, treatmen

Caffeic Acid Induced Apoptosis in MG63 Osteosarcoma Cells Through Activation of Caspases

Background: Caffeic acid has been reported that when it is combined with all-trans retinoic acid, it can inhibit proliferation activity of SaOS-2 or OSA-01 cells. In addition, caffeic acid merely could reduce cell viability of SaOS-2 cells. However, there is not any study in caffeic acid's possible effect to induce apoptosis in osteosarcoma cell.Materials and Methods: MG-63 cells were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum. Cells were treated with various concentrations of caffeic acid. Apoptosis were analyzed with Sub-G1 assay and activation of caspase-8, -9, and -3 were analyzed with immunoblotting. Caffeic acid-induced percentage of apoptotic cells and cleaved-8, -9, -3 were then statistically analyzed.Results: Sub-G1 results showed that caffeic acid significantly induced apoptosis in MG-63 osteosarcoma cells in concentration dependent manner. Immunoblotting results showed that caffeic acid induced cleavage of caspase-8, -9 and -3. Cleaved-caspase-8 and -9 were increased at 1-hour treatment of caffeic acid, while cleaved-caspase 3 was increased markedly at 6-hours treatment of caffeic acid.Conclusions: Caffeic acid induces apoptosis significantly in concentration dependent manner through caspase-dependent intrinsic apoptotic pathway.Keywords: caffeic acid, osteosarcoma, MG-63, apoptosis, caspas

RANKL and TNF-α-induced JNK/SAPK Osteoclastogenic Signaling Pathway was Inhibited by Caffeic Acid in RAW-D Cells

Caffeic acid, a natural substance found majorly in fruits, grains, and herbs, is known to have therapeutic benefits. One of which is to inhibit bone resorption by targeting osteoclastogenesis through inhibition of the Cathepsin K, p38 Mitogenactivated Protein Kinase (MAPK), Nuclear Factor of Activated T-cells c1 (NFATc1) and Nuclear Factor kB (NFkB). Besides p38 MAPK, the c-Jun N-terminal kinase (JNK)/stressactivated protein kinases (SAPK), another member of MAPK family, has been reported to play important roles in osteoclastogenesis. Hence, current study was undertaken in order to investigate mechanism of Caffeic Acid towards JNK/SAPK pathway. Tartrate Resistant Acid Phosphatase (TRAP) staining was performed on caffeic acid-treated and RANKL-TNFα-induced RAW-D cells. Western blot analysis was performed to detect JNK/SAPK and phosphorylated-JNK/SAPK. Protein bands were quantified and statistically analyzed. Treatment of 10 μg/mL Caffeic Acid inhibited 20 ng/mL RANKL and 1 ng/mL TNFα-induced RAW-D differentiation into TRAP+ osteoclast-like polynuclear cells. Induction of 20 ng/mL of RANKL and 1 ng/mL of TNFa for 0.2 or 1 hour, significantly increase phosphorylation of JNK/SAPK as compared with control. Treatment of 10 μg/mL Caffeic Acid significantly inhibited the 20 ng/mL of RANKL and 1 ng/mL of TNFa-induced phosphorylation of JNK/SAPK. Taken together, Caffeic Acid could inhibit the RANKL and TNFa-induced osteoclastogenesis through JNK/SAPK.Keywords : Caffeic Acid, RANKL, TNF, RAW-D cells, osteoclastogenesis, JNK, SAP

Targeting Ameloblatoma into Apoptosis

BACKGROUND: Generally ameloblastoma is a locally aggressive, slow growing, non-metastatic epithelial odontogenic benign tumor. However, rarely some ameloblastoma can metastasize in spite of a benign histologic appearance. Targeting ameloblastoma by inducing it into apoptosis could be a beneficial strategy, since many ameloblastoma cases were reported recurrent after surgical therapy.CONTENT: To investigate ameloblastoma in cellular aspect,cytological pattern of ameloblastoma was divided intoouter layer/peripheral and inner layer/central cells. Tumor necrosis factor (TNF)-α, Fas ligand (FasL), TNF receptor (TNFR)1/death receptor (DR)1, TNFR2/DR2, DR4, DR5andFas were highly expressed in central than peripheral cells. Despite inducing apoptosis, TNF-α can induce PI3K leading to Akt and p44/42 mitogen-activated protein kinases (MAPK) activation in AM-1 cells, which later induce cell survival and proliferation. Therefore apoptotic induction in ameloblastoma should be suggested in higher TNF-α concentration. Expression of FasL and Fas are closely associated with squamous metaplasia and  granular transformation of the tumor cells, suggesting that apoptosis induced by FasL may play a role in the terminally differentiated or degenerative ameloblastoma cells. TNF-related apoptosis-inducing ligand (TRAIL) has emerged as an apoptotic inducing anticancer agent in tumor cells specifically. TRAIL induced activation of caspases, lowering mitochondrial membrane potential, high number of apoptotic cells in ameloblastoma cells. Therefore, TRAIL could be a potential agent for targeting ameloblastoma, although further study should be explored.SUMMARY: Targeting ameloblastoma by inducing it into apoptosis could be achieved effectively, although some criteria should be considered. Therefore understanding the underlying apoptosis signaling pathways are necessary for inducing ameloblasotma into apoptosis. Investigations on other apoptosis-related molecules, potential apoptosis-inducing natural products, and novel approach in reprogramming, are important in the future for a better anagement of ameloblastoma.KEYWORDS: ameloblastoma, apoptosis, TNF, Fas, TRAIL, Akt, MAPK, caspas

Role of Herbal Extract in Stem Cell Development

Stem cell research has been developed and today we can witness some stem cell clinical trials are on going in Indonesia. To meet a successful stem cell treatment, several factors need to be considered, such as cell number. Cell number has been reported to be crucial, and therefore optimal cell number should be achieved. Meanwhile, in some circumstances, cell number is not enough, therefore, cell number should be enriched in an in vitro stem cell culture setting. In an in vitro stem cell culture, besides suitable and sterile equipment, reagents such as culture medium, serum and antibiotics are all important. Although all those criteria are fulfilled, somehow stem cell enrichment cannot be achieved, cell number is still below the target. Modification of stem cell microenvironment should then be an alternative. The addition of growth factors is a part of the strategies to reach a better enrichment. So that, stem cells could later be induced to proliferate at a higher rate. This strategy was then pursued by the scientist involved in herbal medicine. Herbal extracts were now highly investigated due to its potential to induce cell proliferation. Some herbal extracts inducing proliferation and differentiation of stem cell will be shown and described.Keywords: herbal extract, stem cell, progenitor cell, proliferation, differentiatio

Pengaruh Inovasi Produk, Harga, dan Lokasi terhadap Keputusan Pembelian pada Kedai Mie Jontor di Jl. Raya Sawen Tanjung Gresik

Penelitian ini bertujuan untuk mengetahui apakah ada pengaruh Inovasi produk, Harga, dan Lokasi terhadap Keputusan Pembelian pada Kedai Mie Jontor di Jl. Raya sawen Tanjung Gresik. Penelitisn ini  menggunakan jenis kuantitatif. Populasi penelitian ini yaitu konsumen kedai Mie Jontor di Jl. Raya Sawen Tanjung. Teknik pengambilan sampel menggunakan metode accidental sampling. Jumlah sampel sebesar 105 responden. Metode analisis data yang digunakan adalah uji validitas, uji reliabilitas, uji asumsi klasik, analisis regresi linear berganda, uji t, dan uji F. Berdasarkan hasil dari penelitian ini adalah inovasi produk, harga, dan lokasi secara parsial dan simultan berpengaruh terhadap keputusan pembelian. Dapat disimpulkan dalam penelitian ini bahwa inovasi produk yang bagus, harga terjangkau serta lokasi yang bagus akan meningkatkan keputusan pembelian. Maka dengan menciptakan inovasi produk yang baru, harga yang stabil dan terjangkau serta lokasi yang strategis pelaku usaha dapat mencapai tujuan yang diharapakan