Endah Puji Septisetyani, Endah Puji
Research Center for Biotechnology Indonesian Institute of Sciences Jl. Raya Bogor KM 46, Cibinong, Bogor, Indonesia 16911

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OPTIMIZATION OF SODIUM DODECYL SULPHATE AS A FORMAZAN SOLVENT AND COMPARISON OF 3-(4,-5-DIMETHYLTHIAZO-2-YL)-2,5-DIPHENYLTETRAZOLIUM BROMIDE (MTT) ASSAY WITH WST-1 ASSAY IN MCF-7 CELLS Septisetyani, Endah Puji; Ningrum, Ratih Asmana; Romadhani, Yulaika; Wisnuwardhani, Popi Hadi; Santoso, Adi
INDONESIAN JOURNAL OF PHARMACY Vol 25 No 4, 2014
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (871.837 KB) | DOI: 10.14499/indonesianjpharm25iss4pp245

Abstract

A 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay  is  a method that used to measure cell viability. It is based on the conversion of MTT by succinic dehydrogenase enzyme into insoluble formazan. Dissolution of formazan by using proper solvent is the most important step of MTT assay to obtain valid and reliable data. In this study, we observed several solvents [isopropanol, dimethyl sulfoxide (DMSO), and sodium dodecyl sulphate (SDS)] to validate MTT assay by using MCF-7 cells. The observation was performed by MTT addition at concentration of 0.5µg/µL, 3-4h cells incubation at 37°C, dissolution of formed formazan crystal and absorbance measurement at 570nm. The result showed that formazan completely dissolved in DMSO and 10% SDS. The most advantage of using SDS was it avoided the removal of partially dissolved formazan. In this observation, we also found that pH was a very important factor in SDS solution that affected the reaction. The use of optimal condition on MTT assay by SDS-0.01M HCl and SDS-0.025M HCl as formazan solvents showed that IC50 of curcumin were 32.3±0.78µM and 24.08±1.72µM respectively, while WST-1 assay resulted IC50 of curcumin 80.69±5.35µM. Altogether, this study strongly indicated that SDS-0.01M HCl was the best formazan solvent for MTT assay.
n-Butanolic fraction of endofitic fungi of Buah Makasar increases apoptotic effect of doxorubicin on MCF-7 cells Meiyanto, Edy; Kumala, Shirly; Septisetyani, Endah Puji
INDONESIAN JOURNAL OF PHARMACY Vol 20 No 1, 2009
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (664.738 KB) | DOI: 10.14499/indonesianjpharm0iss0pp42-47

Abstract

Makassar fruit, Brucea javanica (L.) Merr., showed chemopreventive activity. Secondary metabolites come from B. javanica fruit, brucatol and bruceantine, induced cell differentiation and apoptosis on Leukemia cell, while quassinoid and its derivates acted as antitumor promoter. Butanolic fraction of supernatan of endofitic fungi 1.2.11 isolate fermentation which isolated from B. javanica fruit showed cytotoxicity toward several cancer cells. This fraction has been predicted contain secondary metabolites from B. javanica and has been identified as Bruceosin and Canthin-6-one derivates. Butanolic fraction (FB) of supernatan from endofitic fungi 1.3.11 isolate fermentation is predicted for having similiar cytotoxycity as active as 1.2.11 isolate. This research is aimed to explore cytotoxycity potention dan doxorubicin on MCF-7 breast cancer cell.Synergism of BF-doxorubicin combination detect from cell viability inhibition and apoptosis induction on MCF-7, a breast cancer cell lines which shows resistancy toward doxorubicin. Cell viability on single treatment of FB and doxorubicin and its combination were carried out by MTT assay to determine IC50 and combination index (CI). Apoptosis induction of FB, doxorubicin and its combination were carried out by ethidium bromideacridine orange DNA staining.n- Butanolic fraction and doxorubicin showed cell viability inhibition on MCF-7 cell with IC50 48 μg/mL and 148 nM, respectively. Both of FB and doxorubicin showed apoptosis induction on IC50. Combination of FBdoxorubicin showed synergism and increased apoptosis induction on MCF-7 cell.Key words: Brucea javanica, endofitic fungi, MCF-7 cell, synergism, doxorubicin.
EXPRESSION OF RECOMBINANT HUMAN ERYTHROPOIETIN WITH GLYCOSYLATION MODIFICATION IN HEK293T CELLS Septisetyani, Endah Puji; Rubiyana, Yana; Wisnuwardhani, Popi Hadi; Wardiana, Andri; Santoso, Adi
INDONESIAN JOURNAL OF PHARMACY Vol 23 No 3, 2012
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (807.905 KB) | DOI: 10.14499/indonesianjpharm0iss0pp177-182

Abstract

Stability  of  erythropoietin  (EPO)  depends  on  its glycosylation  states.  With  more  glycosylation  sites,  the  EPO protein  will  be  more  stable  and  also  increase  its  half-life.  A construct  of  recombinant  human  erythropoietin  (rhEPO)  which contains 2 additional N-link for glycosylation were designed. Based on translation analysis using ORF (open reading frame)-finder and protein  alignment  analysis  using  blast-p  of  NCBI  home  page, expected  recombinant  hEPO  with  additional  6-histidin  tag  in carboxyl terminus  was expressed. HEK293T cells  were transfected with  recombinant  plasmid  containing  rhEPO  by  using  calcium phosphate method. Expression of rhEPO was detected by dot blot and  Western  blot  analysis  using  hEPO  antibody  as  the  primary antibody  and  antirabbit  antibody  with  alkaline  phospatase  linked as  the  secondary  antibody.  The  bands  were  detected  by BCIP/NBT color  development  substrate.  The  data  indicated detection of EPO in culture medium of transfected HEK293T cells.Key  words:  HEK293T  cell,    calcium    phosphate    transfection,  N-linked glycosylation, recombinant human erythropoietin
Optimizaton of Cationic Lipid Mediated Transfection of pEGFP-c1 and pJ-EPO Plasmids in Chinese Hamster Ovary (CHO) Cells Attached Culture for Transient and Stable Recombinant Human Erythropoietin (rhEPO) Expression Septisetyani, Endah Puji; Kusumawati, Arizah; Santoso, Adi
ANNALES BOGORIENSES Vol 19, No 1 (2015): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ab.v19i1.84

Abstract

Cationic lipid is one of transfection agents which show high efficiency and low cytotoxicity. The transfection efficiencies may depend upon the type or amount of cationic lipids, the cell line or DNA plasmid being used for transfection. The purpose of this study was to find optimal condition for transfection of CHO-K1 and CHO-S cells with pJ-EPO plasmid (contain human epo gene) compared with pEGFP-c1 plasmid (contain gfp gene) by cationic lipid Lipofectamin 2000TM to generate stable transfectant expressing recombinant human erythropoietin (rhEPO). Optimization was carried out regarding the amount of lipofectamin, DNA concentration, and concentration of antibiotic Geneticin (G418) for selection of stable transfectants. Using standard amount of lipofectamin (10µl/well) in 6-well plate, highest expression level of green fluorescent protein (GFP) was shown after transfection of CHO-K1 cells with 4µg/well pEGFP-c1 while highest expression level of rhEPO was observed after transfection of CHO-K1 cells with 6, 8, and 10µg/well pJ-EPO plasmids. The data also indicated that optimal transfection conditions of CHO-K1 and CHO-S cells with pJ-EPO were shown with the use of 4µg/well DNA and 15µl lipofectamin, respectively. Concentration of G418 affected the expression where strongest rhEPO expression was shown at 1,000 ng/µl G418 concentration. 
Sequential Adaptation in Mammalian CHO-K1 Cells Producing Human Erythropoietin Wisnuwardhani, Popi Hadi; Septisetyani, Endah Puji; Santoso, Adi
ANNALES BOGORIENSES Vol 21, No 1 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (289.139 KB) | DOI: 10.14203/ab.v21i1.282

Abstract

The production of recombinant proteins for clinical applications using mammalian cell technology has become a prevalent system because of its capacity in assembling functional proteins.  One of the main problems with CHO-K1 cells is that this cell has to grow in the presence of serum. However, the presence of serum will complicate the downstream step for protein production. Thus, protein produced in media without serum, theoretically, would be easier to purify.  Technically, this type of cell can be produced by growing the CHO-K1 cells in serum-free media by using adaptation method in suspension condition. This research showed that through sequential adaptation using conditioned media, the CHO-K1 cell line that produces the human erythropoietin gene (hEPO) was able to grow in suspension culture using serum-free media.  Based on Western blot analysis, it showed that the protein (hEPO) was able to be expressed in suspension culture with molecular mass of about 47 kDa.
8-HIDROKSIISOKAPNOLAKTON-2',3'-DIOL, KUMARIN BIOAKTIF DARI Micromelum minutum Susidarti, Ratna Asmah; Rahmani, Mawardi; Sukari, Mohd. Aspollah; Ali, Abdul Manaf; Mustofa, .; Yasmina, Alfi; Handayani, Sri; Mintarsih, Betty; Ikawati, Muthi’; Septisetyani, Endah Puji
JFIOnline | Print ISSN 1412-1107 | e-ISSN 2355-696X Vol 4, No 3 (2009)
Publisher : Indonesian Research Gateway

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Abstract

Separation of leaves chloroform extract of Micromelum minutum (Rutaceae) yielded a new coumarin, 8-hydroxyisocapnolactone-2¢,3¢-diol. The structure of this compound was characterized by UV, IR, MS and NMR spectroscopic methods, including 1H, 13C, HSQC, COSY, HMBC dan NOESY experiments. This compound is significantly toksisk towards several cancer cell lines (CEM-SS, HL60, HeLa, HepG2, MCF7, T47D and NS1), active against chloroquin sensitive (D10) and resistance (FCR3) Plasmodium falciparum and showed strong antibacterial activity against Bacillus Subtilis mutan, Bacillus. Subtilis wild type, Pseudomonas Aeruginosa dan Staphylococcus aureus resisten meticilin).  ABSTRAK Pemisahan ekstrak kloroform daun Micromelum minutum (Rutaceae) menghasilkan suatu kumarin baru, 8-hidroksiisokapnolakton-2΄,3΄-diol yang strukturnya diidentifikasi secara spektroskopi UV, IR, MS dan NMR termasuk 1H, 13C, HSQC, COSY, HMBC dan NOESY. Senyawa tersebut secara signifikan toksik terhadap beberapa sel kanker (CEM-SS, HL60, HeLa, HepG2, MCF7, T47D dan NS1), aktif terhadap Plasmodium falciparum yang sensitif (D10) maupun resisten (FCR3) kloroquin dan mempunyai aktivitas antibakteri yang kuat terhadap Bacillus Subtilis mutan, Bacillus. Subtilis wild type, Pseudomonas Aeruginosa dan Staphylococcus aureus resisten meticilin).
Synergistic Effect of Areca catechu L. Ethanolic Extract and Its Chloroform Fraction with Doxorubicin on MCF7 MEIYANTO, EDY; HANDAYANI, SRI; SEPTISETYANI, ENDAH PUJI; SUSIDARTI, RATNA ASMAH
JURNAL ILMU KEFARMASIAN INDONESIA Vol 7 No 1 (2009): JIFI
Publisher : Fakultas Farmasi Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1350.634 KB)

Abstract

Ekstrak etanol biji buah pinang (Areca catechu L.) menunjukkan efek sitotoksik pada sel kanker MCF7 dan WiDr. Penelitian ini bertujuan untuk mempelajari efek sinergisme antara ekstrak etanol biji buah pinang (AE) dan fraksi kioroforrnnya (ACF) dengan doxorubicin (Dox) dalam pemacuan apoptosis sel MCF7. Ekstrak etanol dipartisi dengan n-heksan dan kloroform untuk mendapatkan fraksi kloroforin. Efek sitotoksik.AE, ACF, dan Dox pada perlakuan tunggal dan kornbinasi ditentukan dengan metode MTT. Penganiatan apoptosis dilakukan dengan pengecatan DNA dengan akridin oranye-etidium bromida (double staining). Imunositokimia dilakukan untuk melihat ekspresi COX-2 dan Bax. Kombinasi Dox 100, 250, 334, dan 500 nM dengan AE 8 µg/ml; Dox 100 nM dengan AE 20 µg/ml; serta Dox 100 dan 250 nM dengan ACF 7 dan 18 µg/ml memperlihatkan efek sinergistis yang kuat (CI 0,1-0,3). Sernentara itu, kombinasi Dox 250, 334, dan 500 nM dengan AE 20, 27, dan 40 µg/ml; Dox 100 nM dengan AE 27 dan 40 µg/ml; Dox 100 nM dengan AE 20 µg/mi; Serta 500 nM dengan ACF 24 dan 35 µg/ml menunjukkan efek sinergistis (CI 0,3-0,7). Secara keseluruhan, kombinasi AE dan ACF dengan Dox memperiihatkan efek sinergistis pada MCF7 dengan indeks kombinasi (CI) kurang dari 0,9 (P<0,05). Periakuan kombinasi juga memacu apoptosis. Penekanan ekspresi Bcl-2 terjadi padaperlakuan kombinasi ACF-Dox. Hasil penelitian ini menunjulckan bahwa koinbinasi AE dan ACF dengan Dox memberikan efek sinergistis dalam pemacuan apoptosis dengan kemungkinan mekanisme meialui penekanan ekspresi Bcl-2.
Pentagamavunone-0 (PGV-0), a Curcumin Analog, Enhances Cytotoxicity of 5-Fluorouracil and Modulates Cell Cycle in WiDr Colon Cancer Cells Ikawati, Muthi; Septisetyani, Endah Puji
Indonesian Journal of Cancer Chemoprevention Vol 9, No 1 (2018)
Publisher : Indonesian Society for Cancer Chemoprevention

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14499/indonesianjcanchemoprev9iss1pp23-31

Abstract

The use of 5-fluorouracil (5-FU) in colon cancer as the primary chemotherapy has not been meet satisfactory effectiveness.  Therefore, the development of new chemicals as a chemopreventive agent and a combination agent (co-chemotherapeutic agent) for colon cancer is important.  Pentagamavunone-0 (2,5-bis-(4'-hydroxy-3'-methoxybenzylidine) cyclopentanone) (PGV-0), one of curcumin analogs, exhibits cytotoxic effect and apoptosis induction in various cancer cell lines, including colon cancer cell, better than curcumin.  This study aimed to investigate the cytotoxic potency of PGV-0 in combination with 5-FU and their effects, in single or in combination, on cell cycle toward WiDr colon cancer cell line.  The cells were treated with combination concentrations of PGV-0 and 5-FU, and examined by MTT cell viability assay.  The value of combination index (CI) as a parameter of cytotoxic combination assay was measured by a combination index method.  Cells were stained with propidium iodide and the cell cycle distribution was determined by flowcytometry. CI calculation showed additive effects between PGV-0 and 5-FU.  Combination of PGV-0 and 5-FU gave synergism on cell cycle.  Single treatment of PGV-0 increased apoptosis, illustrated as subG1-phase accumulation, stronger than single treatment of 5-FU.  Meanwhile, combination of PGV-0 and 5-FU demonstrated S-phase arrest.  Based on these results, it can be concluded that PGV-0 has the potential to be developed as a co-chemotherapeutic agent for colon cancer but still requires further tracking of its molecular mechanisms.Keywords: Pentagamavunone-0 (PGV-0), 5-fluorouracil (5-FU), colon cancer,combination, cell cycle
Expression of Human Erythropoietin Containing 2 Additional N-Link in CHO-K1 Cells under Different Culture Conditions Santoso, Adi; Larasati, Larasati; Kusumawati, Arizah; Wisnuwardhani, Popi Hadi; Ningrum, Ratih Asma; Septisetyani, Endah Puji
Indonesian Journal of Cancer Chemoprevention Vol 10, No 1 (2019)
Publisher : Indonesian Society for Cancer Chemoprevention

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14499/indonesianjcanchemoprev10iss1pp7-15

Abstract

Human erythropoietin (hEPO) is a glycoprotein that regulates the formation of erythrocytes and mainly used in anemia patients. Previously, we have reported the expression of modified human EPO with 2 additional N-linked in mammalian cell CHO-K1. The aim of this current research was to study the optimum condition for modified recombinant hEPO (rhEPO) production in CHO-K1. To do this, several parameters of culture conditions were applied including antibiotic concentrations, seeding densities, time of incubations, fetal bovine serum (FBS) concentrations and cell culture media. The result showed that the presence of antibiotic G418 improved the expression level with the highest was at 1% of concentration. Meanwhile, seeding density of 2–3x105 cells/6 cm dish and seven day of incubation time were the best condition for rhEPO protein expression. From five different combination media used, F12 medium with 10% FBS gave the highest expression of rhEPO protein. From this study was also found that at passage 16 the expression level was still increasing proving that the clone expressing the protein of our interest is promisingly stable.Keywords : EPO, erythropoietin, protein expression, CHO-K1, optimation
8-HIDROKSIISOKAPNOLAKTON-2',3'-DIOL, KUMARIN BIOAKTIF DARI Micromelum minutum Susidarti, Ratna Asmah; Rahmani, Mawardi; Sukari, Mohd. Aspollah; Ali, Abdul Manaf; Mustofa, .; Yasmina, Alfi; Handayani, Sri; Mintarsih, Betty; Ikawati, Muthiâ??; Septisetyani, Endah Puji
Jurnal Farmasi Indonesia Vol 4, No 3 (2009)
Publisher : Jurnal Farmasi Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.35617/jfi.v4i3.18

Abstract

Separation of leaves chloroform extract of Micromelum minutum (Rutaceae) yielded a new coumarin, 8-hydroxyisocapnolactone-2¢,3¢-diol. The structure of this compound was characterized by UV, IR, MS and NMR spectroscopic methods, including 1H, 13C, HSQC, COSY, HMBC dan NOESY experiments. This compound is significantly toksisk towards several cancer cell lines (CEM-SS, HL60, HeLa, HepG2, MCF7, T47D and NS1), active against chloroquin sensitive (D10) and resistance (FCR3) Plasmodium falciparum and showed strong antibacterial activity against Bacillus Subtilis mutan, Bacillus. Subtilis wild type, Pseudomonas Aeruginosa dan Staphylococcus aureus resisten meticilin).  ABSTRAK Pemisahan ekstrak kloroform daun Micromelum minutum (Rutaceae) menghasilkan suatu kumarin baru, 8-hidroksiisokapnolakton-2Î?,3Î?-diol yang strukturnya diidentifikasi secara spektroskopi UV, IR, MS dan NMR termasuk 1H, 13C, HSQC, COSY, HMBC dan NOESY. Senyawa tersebut secara signifikan toksik terhadap beberapa sel kanker (CEM-SS, HL60, HeLa, HepG2, MCF7, T47D dan NS1), aktif terhadap Plasmodium falciparum yang sensitif (D10) maupun resisten (FCR3) kloroquin dan mempunyai aktivitas antibakteri yang kuat terhadap Bacillus Subtilis mutan, Bacillus. Subtilis wild type, Pseudomonas Aeruginosa dan Staphylococcus aureus resisten meticilin).