Supriatno Supriatno
Department of Oral Medicine, Faculty of Dentistry, Universitas Gadjah Mada

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Antitumor activity of antisense oligonucleotide p45Skp2 in soft palate carcinoma cell squamous in vitro Supriatno Supriatno; Sartari Entin Yuletnawati; Iwa Sutardjo Rus Sudarso
Dental Journal (Majalah Kedokteran Gigi) Vol. 46 No. 1 (2013): March 2013
Publisher : Faculty of Dental Medicine, Universitas Airlangga https://fkg.unair.ac.id/en

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (226.948 KB) | DOI: 10.20473/j.djmkg.v46.i1.p18-22

Abstract

Background: Human soft palate cancers are characterized by a high degree of local invasion and metastasis to the regional lymph nodes. Treatment options for this cancer are limited. However, a new strategy for refractory cancer, gene therapy is watched with keen interest. p45Skp2 gene as a tumor promoter gene is one of target of the oral cancer therapy. To inhibit the activity of p45Skp2 gene is carried-out the genetic engineering via antisense technique. Purpose: To examine the antitumor activity of p45Skp2 antisense (p45Skp2 AS) gene therapy in human soft palate [Hamakawa-Inoue (HI)] cancer cells. Methods: Pure laboratory experimental study with post test only control group design was conducted as a research design. To investigate the apoptosis induction of p45Skp2 AStransfected cell was evaluated by colorimetric caspase-3 assay and Flow cytometry. Furthermore, to detect the suppression of in vitro HI cell invasion and cell growth of p45Skp2 AS-treatment cell was examined by Boyden chamber kit and MTT assay, respectively. Results: The cell number of p45Skp2 AS-treated HI cell was significant decreased when compared with that of p45Skp2 sense (p45Skp2 S) cells (p<0.05). p45Skp2 AS-treated cell induced apoptosis characterized by an increase in the early and late apoptosis, and activation of caspase-3 (p<0.05). Therefore, suppression of HI cell invasion and cell growth were markedly increased by p45Skp2 AS treatment (p<0.05). Conclusion: Antisense oligonucleotide p45Skp2 has a high antitumor activity in human soft palate cancer cell, targeting this molecule could represent a promising new therapeutics approach for this type of cancer.Latar belakang: Kanker palatum lunak mempunyai karakteristik invasi dan metastasis ke limfonodi regional yang tinggi. Pilihan perawatan kanker tersebut masih sangat terbatas. Walaupun demikian, strategi baru untuk penanganan kanker yaitu terapi gen menjadi pilihan utama. Gen p45Skp2 sebagai gen pemacu tumor merupakan salah satu target terapi kanker oral. Untuk menghambat aktivitas gen p45Skp2 tersebut dilakukan rekayasa genetik melalui teknik antisense. Tujuan: Menguji aktivitas antitumor gen p45Skp2 antisense (p45Skp2 AS) terhadap sel kanker palatum lunak (sel HI). Metode: Jenis penelitian yang digunakan adalah eksperimen laboratorik murni dengan rancangan posttest only control group design. Induksi apoptosis sel yang ditransfeksi p45Skp2 AS dievaluasi menggunakan uji caspase-3 kolorimetrik dan flow cytometry. Untuk mendeteksi hambatan invasi dan pertumbuhan sel HI yang ditransfeksi p45Skp2 AS dilakukan uji Boyden chamber dan uji MTT. Hasil: Pertumbuhan sel HI yang ditransfeksi p45Skp2 AS menurun secara signifikan dibandingkan dengan p45Skp2 Sense (S) (p<0,05). Sel HI transfeksi p45Skp2 AS menginduksi apoptosis dengan meningkatkan aktivitas proteolitik caspase-3 dan early and late apoptosis (p<0,05). Hambatan invasi dan pertumbuhan sel HI secara signifikan meningkat pada sel yang diperlakukan dengan p45Skp2 AS (p<0,05). Kesimpulan: p45Skp2 AS oligonukleotida mempunyai aktivitas antitumor yang kuat pada sel kanker palatum lunak. Target dari molekul tersebut dapat menjanjikan suatu terapeutik baru untuk jenis kanker palatum tersebut.
Antitumor activity of intratumoral injection of pcDNA3.1-p27Kip1mt followed by in vivo electroporation in a malignant Burkitt’s lymphoma cell xenograft Supriatno Supriatno; Sartari Entin Yuletnawati
Dental Journal (Majalah Kedokteran Gigi) Vol. 45 No. 4 (2012): December 2012
Publisher : Faculty of Dental Medicine, Universitas Airlangga https://fkg.unair.ac.id/en

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (495.842 KB) | DOI: 10.20473/j.djmkg.v45.i4.p197-201

Abstract

Background: Human malignant Burkitt’s lymphomas are an uncommon type of Non-Hodgkin Lymphoma commonly affects in children. It is a highly aggressive type of B-cell lymphoma. Treatment for this malignant are still limited. However, a new strategy for refractory cancer, gene therapy is watched with keen interest. Recently, a novel method for high-efficiency and region-controlled in vivo gene transfer was developed by combining in vivo electroporation and plasmid cDNA. In the present study, a non-viral gene transfer system, in vivo electroporation in human malignant Burkitt’s lymphoma (Raji) cell xenograft was investigated. Purpose: The purpose of this study was to evaluate p27Kip1 gene therapy in Raji cell xenografts using pcDNA3.1-p27Kip1 mutant type (mt) and pcDNA3.1 empty vector (neo) with the local application of electric pulses. Methods: True experimental study using post-intervention with control group design was performed in this study. Material sample was obtained from integrated research laboratory at faculty of dentistry, Universitas Gadjah Mada, Yogyakarta. The efficiency of transfection of exogenous p27Kip1 gene by electroporation was confirmed by Western bloting analysis. To evaluate the reduction of malignant Burkitt’s lymphoma cell xenografts by this method, the volume of Raji cell xenografts in mice after electroporation with p27Kip1 mt or neo gene was measured. Results: Up-regulation of p27Kip1 protein was detected in pcDNA3.1-p27Kip1 mt. Furthermore, the growth of tumors was markedly suppressed by p27Kip1 mt gene transfection compared with transfection of neo. Conclusion: Injection of pcDNA3.1-p27Kip1 mt gene followed by in vivo electroporation has a high-potentially to suppress the growth of malignant Burkitt’s lymphoma cells. Furthermore, combination system of pcDNA3.1-p27Kip1 mt-injected tumor and electroporation might be used for human oral cancer. Latar belakang: Limfoma Burkitt’s maligna banyak terjadi pada anak-anak dan merupakan jenis yang langka dari limfoma NonHodgkin (NHL). Limfoma Burkitt’s maligna adalah tipe yang sangat agresif dari limfoma sel B. Perawatan penyakit ini masih sangatterbatas, walaupun demikian strategi baru perawatan kanker menggunakan terapi gen menjadi pusat perhatian. Suatu metode baru transfer gen untuk meningkatkan efisiensi dan kontrol area telah dikembangkan dengan mengkombinasi elektroporasi in vivo dan plasmid cDNA. Pada penelitian ini, telah diteliti sistim transfer gen non-virus dengan elektroporasi in vivo terhadap xenograft sel limfoma Burkitt’s maligna (sel Raji). Tujuan: Tujuan dari penelitian ini adalah untuk mengevaluasi terapi gen p27Kip1 terhadap xenograft sel Raji menggunakan pcDNA3.1-p27Kip1 mutant type (mt) dan pcDNA3.1 empty vector (neo) dengan aplikasi lokal elektroporasi. Metode: Jenis penelitian yang digunakan adalah eksperimen murni memakai rancangan pasca intervensi dengan kelompok kontrol. Sampel dan bahan penelitian didapat dari laboratorium riset terpadu, Fakultas Kedokteran Gigi, Universitas Gadjah Mada, Yogyakarta. Efisiensi transfeksi gen p27Kip1eksogen dengan elektroporasi dilakukan dengan analisis Western bloting. Untuk mengevaluasi hambatan xenograft sel limfoma Burkitt’s maligna dengan metode elektroporasi, dilakukan pengukuran volume xenograft sel Raji pada tikus pasca elektroporasi dan injeksi gen p27Kip1 mt atau neo. Hasil: Peningkatan regulasi protein p27Kip1 terdeteksi pada gen pcDNA3.1-p27Kip1 mt. Selanjutnya, pertumbuhan tumor secara signifikan terhambat oleh transfeksi gen p27Kip1 mt dibandingkan dengan transfeksi neo. Kesimpulan: Injeksi gen pcDNA3.1-p27Kip1 mt disertai elektroporasi in vivo mempunyai potensi yang kuat menghambat pertumbuhan "> sel limfoma Burkitt’s maligna. Kombinasi sistim injeksi tumor menggunakan gen pcDNA3.1-p27Kip1 mt dan elektroporasi kemungkinan dapat digunakan untuk terapi kanker oral.
Electro-gene therapy in a human oral tongue cancer cell by intratumoral injection of pcDNA3.1-p27Kip1 wt Supriatno Supriatno
Dental Journal (Majalah Kedokteran Gigi) Vol. 40 No. 1 (2007): March 2007
Publisher : Faculty of Dental Medicine, Universitas Airlangga https://fkg.unair.ac.id/en

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (448.145 KB) | DOI: 10.20473/j.djmkg.v40.i1.p1-5

Abstract

Oral tongue cancers are characterized by a high degree of local invasion and a high rate of metastases to the cervical lymph nodes. Also, treatment options for this cancer are limited. However, a new strategy for refractory cancer, gene therapy is watched with keen interest. Recently, a novel method for high-efficiency and region-controlled in vivo gene transfer was developed by combining in vivo electro-gene therapy and intratumoral plasmid DNA injection. In the present study, a nonviral gene transfer system, in vivo electrogene therapy in human oral tongue cancer cell, SP-C1 xenograft was examined. The aim of the study is to examine the efficiency of transfection of exogenous p27Kip1 gene by electroporation and the antitumor activity of p27Kip1 gene therapy in human oral tongue cancer xenografts using pcDNA3.1-p27Kip1 wild type (wt) and pcDNA3.1 empty vector with the local application of electric pulses. To evaluate this in vivo gene transfer method, the enhanced green fluorescence protein (EGFP) gene was transfected into xenografts by electroporation. The efficiency of transfection of exogenous p27Kip1 gene by electroporation was confirmed by Western blotting analysis. To estimate the reduction of oral tongue cancer xenografts by this method, the size of SP-C1 xenografts in nude mice after electroporation with wild type p27Kip1 gene was measured. The growth of tumors was markedly suppressed by wild type p27Kip1 gene transfection by electroporation compared with transfection of empty vector only. Moreover, histological specimens revealed apoptotic cell death was increased in wild type p27Kip1-transfected tumors than empty vector. These results suggest that it is possible to transfer wild type p27Kip1 into human oral tongue cancer xenografts using electroporation. Wild type p27Kip1 has a high-potencially to suppress the growth of tumors. Finally, combination system of pcDNA3.1-p27Kip1 wt-injected tumor and electroporationmight be used for human oral cancer.