Cell Morphological Change and Caspase-3 Protein Expression on Epithelial Cells under Stimulation of Oral Bacterium Streptococcus sanguinis

Oral commensal bacterium Streptococcus sanguinis may find in periodontal lesions, deep seated infection, and infective endocarditis that are usually dominated by anaerobes. This bacterium caused cell death on some cells but host responses to this species remained unclear. Objective: This study was aimed to detect cell morphologica change and role of caspase-3 in cell death mechanism induced by S. sanguinis. Methods: HeLa cells as representative model for oral epithelial cells were exposed to 107 cells/ml bacteria for 48 h. Morphological change was observed microscopically after hematoxyline-eosin staining. Expression of active caspase-3 was examined by immunocytochemical analysis after cell stimulation for 36 and 48 h with wild type supragingival S. sanguinis. Doxorubicin (0.5625 μg/ml) was used as positive control for caspase-3 activation. Results: The results showed cell shrinkage of bacterial-treated cells; and active caspase-3 molecules were detected after 36 and 48 hours cell stimulation. Conclusion: This study would suggest cell shrinkage and caspase-3-dependent apoptotic cell death induced by S. sanguinis.DOI: 10.14693/jdi.v22i1.37

Caspase-3-dependent Cell Death in B lymphocyte Caused by Pseudomonas aeruginosa Pyocyanin

Objective: The aim of this study was to investigate cellular responses of B lymphocyte to the exposure of pyocyanin and the role of caspase-3 in its molecular mechanism. Methods: B lymphocytes (Raji cells) were cultured overnight prior to the experiments. Cell culture in five replications were then exposed to various concentrations of pyocyanin and incubated for 24 h in antibiotics-free medium. MTT assay was performed to analyze the cytotoxicity effect of pyocyanin. In separated experiments, the cells were cultured with pyocyanin and addressed for cell morphological analysis using phase contrast microscope. To study the mechanism involved in pyocyanin-induced cellular damage, immunocytochemical analysis was run for the identification of active caspase-3 protein expression. Results: The results of this study showed that cell viability was decreased in pcyocyanin-treated groups. Statistical analysis using ANOVA (p < 0.05) demonstrated significant different between groups with significant value of 0.000. Pyocyanin induced cell death on B lymphocyte in dose-dependent manner. Nuclear fragmentation was observed in pyocyanin-induced cell death; furthermore, caspase-3 was expressed clearly in cell cytoplasm after 24 h incubation. Conclusion: It is concluded that pyocyanin is capable of inducing cell death on B lymphocyte. Caspase-3 may play important role in the molecular mechanism of pyocyanin-induced cell death.DOI: 10.14693/jdi.v22i2.40

Perubahan morfologi sel HeLa setelah paparan ekstrak etanolik Curcuma longa

Cell morphological changes on HeLa cells after Curcuma longa etanolik extract exposure. Curcuma is mostly found in the areas with tropical and sub-tropical climate, and is one of original plants of Southeast Asia. In Indonesia, curcuma can be found in almost all regions and areas. Curcumin, which is curcuma’s main constituent, is a potent anti oxidant. Previous studies reported that curcuma longa extract may decrease the growth of cancer cells by interfering with cell proliferation, and by causing the cell apoptosis; however, the mechanism of apoptosis remains unclear. The purpose of this study was to investigate the effect of Curcuma longa extract on the morphological change of HeLa cells, indicating the cell damage. HeLa cells (5x10⁴ cells/well) were cultured in complete RPMI 1640 overnight before stimulation. Etanol extract of Curcuma longa (50 µg/ml, 100 µg/ml, 150 µg/ml) were added to the culture of HeLa cells and were incubated for 24 hours in antibiotic-free of culture medium. HeLa cells morphological analysis was performed under phase contrast microscope after haematoxilent eosin staining. Docsorubisin (0,5625 mg/ml) was used as positive control in this study. The results demonstrated that Curcuma longa extract caused cell morphological changes on HeLa cells indicated by cell shrinkage, lost contact with neighboring cells as the alteration of apoptotic cell death in most of cell population. The nuclei were dark as a result of their capability to absorb haematoxylene dye. Statistical analysis showed a significant difference between controls and treatment groups. It was then concluded that Curcuma longa extract induced cell damage on HeLa cells in a way of cell shrinkage.ABSTRAKKunyit (Curcuma longa) merupakan tanaman yang dapat tumbuh di daerah tropis dan sub tropis, serta merupakan tanaman asli Asia Tenggara. Di Indonesia, kunyit menyebar secara merata di seluruh daerah. Kurkumin yang merupakan unsur utama kunyit, merupakan antioksidan yang kuat. Beberapa penelitian juga menunjukkan bahwa kunyit mampu menghambat pertumbuhan beberapa tipe sel kanker. Mekanisme anti-kanker kurkumin adalah dengan menghambat proliferasi sel. Penelitian terdahulu melaporkan bahwa ekstrak Curcuma longa menginduksi apoptosis pada sel HeLa, tetapi mekanisme kematian sel tersebut belum jelas. Tujuan penelitian ini adalah untuk mengkaji pengaruh ekstrak Curcuma longa pada perubahan morfologi sel HeLa, dimana perubahan morfologi merupakan parameter kerusakan sel. Sel HeLa (5x104 sel/well) dikultur dalam RPMI 1640 semalam sebelum stimulasi. Ekstrak etanol kunyit (50 µg/ml, 100 µg/ml, 150 µg/ml) ditambahkan pada kultur HeLa dan diinkubasi selama 24 jam dalam medium tanpa antibiotik. Analisis morfologi sel HeLa dilakukan dengan menggunakan mikroskop fase kontras setelah pewarnaan haematoksilen eosin. Doksorubisin (0,5625 µg/ml) digunakan sebagai kontrol positif induksi apoptosis. Hasil penelitian menunjukkan bahwa ekstrak Curcuma longa menyebabkan perubahan morfologi sel yang ditandai dengan semakin mengecilnya ukuran sel, hilangnya prosesus sitoplasmik sehingga sel berbentuk bulat, serta hilang kontak dengan sel lain yang merupakan ciri apoptosis pada sebagian besar sel HeLa. Nukleus tampak berwarna gelap karena peningkatan kapasitas penyerapan zat haematoksilen. Analisa statistik menunjukkan perbedaan yang bermakna antara kelompok kontrol positif dan negatif dengan kelompok stimulasi dalam jumlah sel yang mengalami perubahan morfologi menuju apoptosis. Disimpulkan bahwa ekstrak Curcuma longa mampu menginduksi perubahan morfologi sel HeLa yaitu berupa cell shrinkage

Ekspresi Caspase-3 Pada Sel Epitel Rongga Mulut (Kb Cell Line) Setelah Paparan Ekstrak Kopi

Kopi adalah minuman yang biasa dikonsumsi oleh masyarakat sehari-hari. Telah diketahui bahwa kopi mengandung kafein seperti yang terdapat juga pada teh dan coklat. Kandungan terbanyak kafein terdapat pada kopi. Kafein mempunyai struktur kimia 1, 3, 7- trimethylxanthine dan merupakan derivat xanthine. Senyawa ini dapat menginduksi kematian sel yang mengarah pada apoptosis, namun mekanisme yang terlibat belum diketahui dengan jelas. Tingginya konsumsi kopi di dunia yang selalu meningkat mengindikasikan perlunya dilakukan penelitian untuk mengetahui efek kafein pada epitel rongga mulut yang berkontak langsung dengan kafein. Penelitian terdahulu melaporkan bahwa ekstrak kopi menyebabkan kerusakan sel yang sebagian besar mengarah pada apoptosis, tetapi mekanismenya belum jelas. Tujuan penelitian ini adalah untuk menganalisis mekanisme kematian sel KB yang diinduksi oleh kafein melalui aktivasi caspase-3. Sel KB sebagai model epitel oral (5x10⁴sel) dikultur dalam DMEM menggunakan 24 wells microplate selama 24 jam sebelum perlakuan. Sel selanjutnya dipapar dengan kafein dengan konsentrasi 100 µg/ml, 200 µg/ml, 400 µg/ml dan diinkubasi selama 24 dan 48 jam dalam DMEM. Doxorubicin (0,5625 µg/ml) digunakan sebagai kontrol positif induksi apoptosis. Teknik imunositokimia terhadap caspase-3 dilakukan pada sel setelah dipapar kafein untuk mengamati adanya ekspresi caspase-3 sebagai ciri apoptosis. Identifikasi caspase-3 dilakukan menggunakan mikroskop fase kontras. Ekspresi protein caspase-3 terdeteksi pada sitoplasma sel KB. Hasil penelitian ini menunjukkan adanya ekspresi caspase-3 aktif yang ditandai dengan warna cokelat dengan intensitas kuat pada sitoplasma sebagian besar sel setelah dipapar kafein dengan konsentrasi 100 μg/ml dan 200 µg/ml selama 24 jam. Disimpulkan bahwa ekstrak kopi menyebabkan apoptosis sel KB melalui jalur aktivasi caspase-3. The Expression of Caspase-3 in Oral Cavity (Kb Cell Line) after Exposure to Coffee Extract. People widely consume coffee in daily meals. It is known there is caffeine found in coffee like it is found in tea and chocolate. Caffeine is found in the greatest amount of coffee. This 1, 3, 7- trimethyl xanthine substance is a derivate of xanthine that is consumed by almost all people in the world. This substance could induce cell death that mainly is apoptosis, but how the mechanism has not been clearly understood. Considering that coffee is widely consumed in the whole world, it is necessary to conduct an experiment to find any possible effect of caffeine to oral epitel that make direct exposure to caffeine. This experiment is targeted to analyze the mechanism of cell death which caused by caffeine through activation of caspase-3. KB cells as oral epithelial model (5x1044 sel) were cultured in DMEM using 24 well microplate for 24 hours before treatment. Then caffeine was given with concentration of 100 µg/ml, 200 µg/ml and 400 µg/ml. Cells were then incubated for 24 and 48 hours period in DMEM. Doxorubicin (0,5625 µg/ml) was used as a positive control of apoptosis induction. Immunocytochemistry technique was then done to observe any caspase three expression as a marker for apoptosis. Identification of active caspase-3 was then done using contrast phase microscope. The results showed expression of caspase-3 in KB cells cytoplasm which observed as high intensity of brown colored molecules in cell cytoplasm after 100 μg/ml and 200 µg/ml caffeine exposure in 24 hours. It was concluded that coffee extract induce KB cells apoptosis through caspase-3 activation mechanism

Aktivitas Penghambatan Candida krusei oleh Ekstrak Etanol Batang Brotowali (Tinospora crispa L.)

Candida krusei merupakan salah satu jenis Candida dengan tingkat patogenitas yang lebih rendah dibandingkan C. albicans. Meskipun demikian spesies ini dapat menyebabkan penurunan respon imun inang karena memiliki struktur polisakarida chitin yang lebih tinggi. Pengobatan infeksi Candida yang disamaratakan mengakibatkan C. krusei menjadi resisten terhadap obat antijamur. Ekstrak etanol batang brotowali (Tinospora crispa L.) mengandung flavonoid dan alkaloid-berberin yang dapat menghambat pertumbuhan jamur. Penelitian ini bertujuan untuk menguji kemampuan ekstrak etanol batang brotowali dalam menghambat pertumbuhan C. krusei. Pembuatan ekstrak etanol batang brotowali dilakukan dengan menggunakan metode maserasi. Uji antijamur dilakukan dengan teknik difusi kertas cakram (Kirby-Bauer test). Ekstrak etanol batang brotowali mampu menghambat pertumbuhan C. krusei pada konsentrasi 2.500 μg/mL dan 5.000 μg/mL dengan zona hambat sebesar 17,75 mm dan 22,25 mm. Pada konsentrasi 1.250 μg/mL, zona hambat yang terbentuk sebesar 6,75 mm yang termasuk ke dalam kategori respon hambat pertumbuhan yang lemah. Analisis statistik menggunakan One Way Anova menunjukkan nila p < 0,05. Kesimpulannya adalah ekstrak etanol batang brotowali mampu menghambat pertumbuhan C. krusei dengan konsentrasi efektif 2.500 μg/mL.Candida krusei merupakan salah satu jenis Candida dengan tingkat patogenitas yang lebih rendah dibandingkan C. albicans. Meskipun demikian spesies ini dapat menyebabkan penurunan respon imun inang karena memiliki struktur polisakarida chitin yang lebih tinggi. Pengobatan infeksi Candida yang disamaratakan mengakibatkan C. krusei menjadi resisten terhadap obat antijamur. Ekstrak etanol batang brotowali (Tinospora crispa L.) mengandung flavonoid dan alkaloid-berberin yang dapat menghambat pertumbuhan jamur. Penelitian ini bertujuan untuk menguji kemampuan ekstrak etanol batang brotowali dalam menghambat pertumbuhan C. krusei. Pembuatan ekstrak etanol batang brotowali dilakukan dengan menggunakan metode maserasi. Uji antijamur dilakukan dengan teknik difusi kertas cakram (Kirby-Bauer test). Ekstrak etanol batang brotowali mampu menghambat pertumbuhan C. krusei pada konsentrasi 2.500 μg/mL dan 5.000 μg/mL dengan zona hambat sebesar 17,75 mm dan 22,25 mm. Pada konsentrasi 1.250 μg/mL, zona hambat yang terbentuk sebesar 6,75 mm yang termasuk ke dalam kategori respon hambat pertumbuhan yang lemah. Analisis statistik menggunakan One Way Anova menunjukkan nila p < 0,05. Kesimpulannya adalah ekstrak etanol batang brotowali mampu menghambat pertumbuhan C. krusei dengan konsentrasi efektif 2.500 μg/mL

Cell Morphological Change and Caspase-3 Protein Expression on Epithelial Cells under Stimulation of Oral Bacterium Streptococcus sanguinis

<p><span> Oral commensal bacterium <em>Streptococcus sanguinis </em></span>may find in periodontal lesions, deep seated infection, and infective endocarditis that are usually dominated by anaerobes. This bacterium caused cell death on some cells but host responses to this species remained unclear. <strong>Objective: </strong>This study was aimed to detect cell morphologica change and role of caspase-3 in cell death mechanism induced by <em>S. sanguinis</em>. <strong>Methods: </strong> HeLa cells as representative model for oral epithelial cells were exposed to 107 cells/ml bacteria for 48 h. Morphological change was observed microscopically after hematoxyline-eosin staining. Expression of active caspase-3 was examined by immunocytochemical analysis after cell stimulation for 36 and 48 h with wild type supragingival S. sanguinis. Doxorubicin (0.5625 μg/ml) was used as positive control for caspase-3 activation. Results: The results showed cell shrinkage of bacterial-treated cells; and active caspase-3 molecules were detected after 36 and 48 hours cell stimulation. <strong>Conclusion:</strong> This study would suggest cell shrinkage and caspase-3-dependent apoptotic cell death induced by<em> S. sanguinis.</em></p><p>DOI: 10.14693/jdi.v22i1.375</p

CELLULAR AND MOLECULAR STUDY OF APOPTOSIS INDUCED BY Streptococcus sanguinis ON HeLa CELLS, IN VITRO STUDY

Streptococcus sanguinis is oral commensal bacterium. It is known that S. sanguinis is aerob-facultative bacteria, but it also found in periodontal lesions and deep abscess that usually dominated by anaerob bacteria. This bacterium is often implicated with the pathogenesis of infective endocarditis. This species can induce cell death on some cells. The purpose of this study was to identify cell morphological change during apoptotic process and to clarify the role of caspase3 in the apoptotic mechanism induced by S. sanguinis in HeLa epithelial cells. HeLa cells were treated with 10� cell/ml S. sanguinis suspension and incubated for 36 and 48 hour

Induksi Kerusakan pada Sel Epitel (HeLa) oleh Bakteri Komensal Oral Streptococcus sanguinis

Abstrak Bakteri komensal oral Streptococcus sanguinis sejauh ini diketahui sebagai spesies yang bertanggung-jawab pada kolonisasi awal bakteri pada permukaan gigi. Bakteri ini secara jelas berperan dalam patogenesis infective endocarditis dan dijumpai pada lesi jaringan periodontal; namun perannya dalam patogenesis infeksi oral masih belum diketahui dengan jelas. Penelitian terhadulu menunjukkan bahwa S. sanguinis mampu menginduksi apoptosis pada sel epitel HeLa, tetapi mekanisme kematian sel tersebut belum jelas. Tujuan penelitian ini adalah untuk mengkaji pengaruh bakteri S. sanguinis pada prubahan seluler dan molekuler nukleus sel HeLa. Sel HeLa (5x105 sel/well) dikultur dalam RPMI 1640 semalam sebelum stimulasi. Suspensi bakteri S sanguinis (107 sel/ml) ditambahkan pada kultur HeLa dan diinkubasi selama 24 dan 48 jam dalam medium tanpa antibiotik. Analisis morfologi sel HeLa dilakukan dengan menggunakan mikroskop fase kontras dan mikroskop fluoresen setelah pewarnaan nukleus Hoechst 33342. Keutuhan DNA pada kelompok kontrol dan stimulasi bakteri diamaati setelah elektroforesis gel agarose. Profil DNA setiap sampel diamati menggunakan UV transiluminator. Doxorubicin (0,5625 mg/ml) digunakan sebagai kontrol positif induksi apoptosis. Hasil penelitian ini menunjukkan bahwa S. sanguinis menyebabkan perubahan morfologi sel, ditandai dengan berkurangnya ukuran sel seperti tampak pada mikroskop fase kontras serta peningkatan intensitas serapan fluoresen nukelus. Degradasi DNA terjadi pada sel yang terpapar S. sanguinis. Disimpulkan bahwa S. sanguinis mampu menginduksi kerusakan pada sel epitel HeLa. Kata kunci: Streptococcus sanguinis, perubahan morfologi sel, kerusakan DNA Abstract An oral commensal bacterium Streptococcus sanguinis is known as species which is responsible for early bacterial colonization within dental plaque. This bacterium has been implicated clearly with the pathogensis of infective endocarditis and some periodontal lession; however, it’s role in the patogenesis of oral infection still unknown. In previous study it was found that S. sanguinis induced apoptotic cell death on epithelium cell line, HeLa cells, but the mechanism still needs to be clarified. Therefore, the aim of this study was to investigate the effect of S. sanguinis whole cell bacteria to cellular and molecular change of HeLa cells nuclei. The cells (5x105 cells/well) were cultured in complete RPMI 1640 medium for overnight before stimulation. Streptococcus sanguinis bacterial suspension (107 cells/ml) was added to the culture and incubated for 24 and 48 hours in antibiotic-free culture medium. HeLa cells morphological analysis was performed under phase contrast and fluorescence microscope observation after Hoechst 33342 staining. In order to observe the integrity of DNA, HeLa whole cell DNA of untreated and bacteria-treated group were isolated addressed for agarose gel electrophoresis. The DNA profil of each sample was observed under UV transilluminator. An apopotic inducer Doxorubicin (0,5625 mg/ml) was used as a positive control in this study. The results demonstrated that S. sanguinis caused cell morphological change on HeLa cells indicated by decrease in cell size as shown on phasecontrast microscopy and increased fluorescence intensity of nucleus. A smear DNA degradation was observed after agarose gel electrophoresis of S. sanguinis-treated cells. It was concluded that S. sanguinis could induce cell damage on HeLa cells. Keywords: Streptococcus sanguinis, cell morphological change, DNA damag