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Abstrak :
ABSTRAK
belakang: Sambiloto Andrographis paniculata Ness. telah digunakan secara luas sebagai obat tradisional. Tanaman ini mempunyai potensi sebagai antikanker. Andrografolida sebagai senyawa aktif utama sambiloto telah terbukti bersifat sitotoksik terhadap berbagai jenis sel kanker. Evaluasi toksisitas penting dilakukan untuk memastikan keamanan andrografolida dan tanaman sambiloto. Penelitian ini bertujuan menganalisis efek andrografolida dan ekstrak/fraksi sambiloto terhadap viabilitas, siklus, serta faktor transkripsi diferensiasi sel punca mesenkimal asal sumsum tulang manusia bone marrow mesenchymal stem cells/BMMSC menjadi osteoblas. Metode: Penilaian efek andrografolida dan fraksi etil asetat sambiloto FEAS terhadap viabilitas, siklus sel serta tingkat ekspresi mRNA CDK4 dan p21 dilakukan dengan memaparkan andrografolida konsentrasi 5, 10 dan 15 g/mL dan FEAS konsentrasi 20, 40 dan 60 g/mL selama 12, 24 dan 36 jam. Viabilitas sel diperiksa berdasarkan prinsip reduksi garam formazan WST-8, analisa siklus sel menggunakan flow cytometer dan tingkat ekspresi mRNA dengan quantitative RT-PCR. Penilaian terhadap diferensiasi osteoblas dilakukan dengan menginduksi BMMSC dengan medium osteogenik disertai pemberian andrografolida konsentrasi 5 dan 10 g/mL serta FEAS konsentrasi 20 dan 40 g/mL selama 7, 14 dan 21 hari, kemudian dinilai intensitas pewarnaan alizarin red AR , kadar deposit kalsium ekstraseluler serta tingkat ekspresi runx2 dan osterik. Hasil: Andrografolida dan FEAS menurunkan viabilitas BMMSC sesuai tingkatan konsentrasi dan waktu paparan. Kedua bahan uji tersebut menghambat proliferasi BMMSC dengan meningkatkan secara bermakna persentase populasi sel pada fase G1 dan menurunkan populasi sel yang memasuki fase S dan G2 siklus sel pada paparan 24 jam. Tidak terdapat efek terhadap tingkat ekspresi mRNA CDK4 namun ekspresi mRNA p21 meningkat bermakna. Andrografolida dan FEAS menurunkan intensitas warna merah AR, kadar matriks kalsium ekstraseluler dan ekspresi mRNA runx2 secara bermakna, namun meningkatkan ekspresi mRNA osterik pada proses diferensiasi BMMSC menjadi osteoblas Kesimpulan: Andrografolida konsentrasi 5, 10 dan 15 g/mL maupun fraksi etil asetat sambiloto konsentrasi 20, 40 da 60 g/mL mempunyai efek toksik terhadap viabilitas, proliferasi dan diferensiasi osteoblas pada BMMSC secara in vitro. Kata kunci : Andrografolida, Andrographis paniculata, BMMSC, siklus sel, osteoblas.

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Background: Sambiloto Andrographis paniculata Ness./AP , has been used extensively as a traditional medicine. Andrographolide as the main active component of this plant showed cytotoxic activity in vitro on various type of cancer cells line. Toxicity evaluation is important to ensure the safety of andrographolide and this bitter plant. The objective of this study was to investigate the effects of andrographolide and the extract of AP on viability, cell cycle, and transcription factors of osteoblast differentiation on human bone marrow mesenchymal stem cells BMMSC Methods: BMMSC were treated with andrographolide at 5, 10 and 15 ?g/mL and ethyl acetate fraction of AP EAFA at 20, 40 and 60 ?g/mL for 12, 24 and 36 hours. The cells viability was assessed using tetrazolium salt WST-8 assay, the cell cycle was evaluated using flow cytometer with propidium iodide DNA?binding fluorescent dyes and the expression of CDK4 mRNA and p21 was analyzed by RT-PCR. Further examination was investigated the effects of the compounds on the osteogenic differentiation of BMMSC. The cells were cultured on osteogenic medium and treated with andrographolide at 5 and 10 ?g/mL and EAFA at 20 and 40 ?g/mL for 7, 14 and 21 days. The matrix mineralization was assessed by alizarin red-s staining AR , the semi-quantification of calcium was determined by acetic acid extraction of calcium binding AR and the expression of runx2 and osterix were analysed by RT-PCR Results: This research was revealed that andrographolide and EAFA decreased the cell viability, arrested the cell cycle at G1 phase, and up regulated the expression of mRNA p21. Moreover andrographolide and EAFA supplementation decreased the intensity of AR and calcium deposition on cell culture. The expression of transcriptor factors runx2 was down regulated while osterix was up regulated. Conclusion: Andrographolide at 20, 40 and 60 ?g/mL and EAFA at 20, 40 and 60 ?g/mL showed potentially toxic on cell viability, arrested cell cycle and impaired osteoblast differentiation of BMMSC in vitro.

Abstrak :

Respons radiasi kanker serviks diduga dapat ditingkatkan dengan pemilihan waktu radiasi tetap yang berpola sirkadian karena dianggap sesuai dengan fase radiosensitif G₂-M sel kanker. Daur sirkadian dan melatonin dianggap berperan dalam radiosensitivitas. Dihipotesiskan respons radiasi pagi hari maupun efek samping radiasi pagi hari akan lebih baik dibanding sore hari.

Penelitian ini merupakan uji klinis dengan perolehan subjek secara berurutan. Alokasi pilihan waktu radiasi pada pagi (06.00–08.00) dan sore (16.00–18.00) hari dengan randomisasi blok tiap enam subjek terpisah antara stadium II dan III. Data diperoleh menggunakan metode open label. Pengukuran data klinis seperti ukuran tumor, respons klinis, dan efek samping dilakukan oleh dua dokter independen yang terlatih. Dilakukan pengukuran kadar melatonin dan fase G₂-M siklus sel di institusi resmi. Respons baik dan buruk ditetapkan berdasarkan kriteria WHO sedangkan efek samping ada atau tidak, ditetapkan berdasarkan kiriteria RTOG.

Penyinaran di waktu pagi menunjukkan respons klinis lebih baik dibandingkan sore (p 0,025; 95% IK:1,27–33,08; adj OR: 6,48) untuk respons pascaradiasi maupun 2–4 minggu pascaradiasi (p 0,048; 95% IK 1,02–47,81; adj OR 6,98). Kadar Hb awal dan ukuran klinis tumor berpengaruh secara bermakna terhadap respons baik pascaradiasi maupun respons baik 2–4 minggu pascaradiasi. Dalam hal efek samping, pilihan waktu radiasi tidak menunjukkan hasil yang bermakna, namun kadar melatonin praradiasi berpengaruh, khususnya efek samping kulit (p 0,006; 95% IK 1,66–18,99; adj OR 5,62). Variabel yang bermakna memengaruhi efek samping terapi pada gastrointestinal adalah overall treatment time (p 0,031; 95% IK 1,19–39,93; adj OR 6,89), sedangkan untuk genitourinaria adalah PA diferensiasi (p 0,015; 95% IK 1,51–46,37; adj OR 8,36), penurunan berat badan (p 0,025; 95% IK 1,22–18,30; adj OR 4,72), dan nyeri sebelum radiasi (p 0,017; 95% IK 1,31–15,32; adj OR 4,47).

Simpulan: Respons radiasi kanker serviks uteri yang diradiasi pagi hari lebih baik daripada yang diradiasi sore hari, namun efek samping radiasi pagi hari tidak berbeda bermakna dibandingkan sore hari. Belum dapat dipastikan pengaruh besarnya proporsi fase G₂-M terhadap respons klinis radiasi. Ada kecenderungan pengaruh kadar melatonin pagi hari terhadap respons klinis radiasi dan terbukti kadar melatonin berpengaruh pada efek samping kulit.

Kata kunci: kanker serviks, melatonin, radiosensitivitas, siklus sel, sirkadian

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The radiation response of cervical cancer can be enhanced by the choice of a fixed radiation time of circadian pattern because it is considered to be in accordance with the radiosensitive phase of G₂-M cancer cells. Circadian cycles and melatonin are thought to play a role in radiosensitivity. It is hypothesized that the response and side effects of morning radiation will be better than the afternoon.

This study was an RCT (randomized clinical trials) with consecutive sampling. Treatment allocation for radiation time in the morning (06.00–08.00) and afternoon (16.00–18.00) were determined by block randomization for every six subjects based on the stage (II and III). The data was obtained with an open label method. Measurement of clinical data such as tumor size, clinical response, and side effects were carried out by two-trained independent physicians. Measurement of melatonin levels and G₂-M phases of cell cycle were carried out in official institution. Good and poor responses were set based on WHO criteria while the side effects were determined based on the RTOG criteria.

Morning radiation showed a better post-radiation and 2–4 weeks post-radiation clinical response compared with afternoon (p 0.025; 95% CI:1.27–33.08; adj OR: 6.48 and p 0.048; 95%CI 1.02–47.81; adj OR 6.98, respectively). The initial Hb level and clinical size of the tumor had a significant effect on good response both post-radiation and 2-4 weeks post-radiation. In regards to the side effects, radiation time did not show significant results in causing side effects, but pre-radiation melatonin level did on skin (p 0.006; 95%CI 1.66–18.99; adj OR 5.62). The significant variable in influencing gastrointestinal side effects was overall treatment time (p 0.031; 95%CI 1.19–3.93; adj OR 6.89), whereas for genitourinaria were differentiation of histopathology (p 0.015; 95%CI 1.51–46.37; adj OR 8.36), weight loss (p 0.025; 95%CI 1.22–18.30; adj OR 4.72), and presence of pain pre-radiation (p 0.017; 95%CI 1.31–15.32; adj OR 4.47).

Conclusion: The radiation response of irradiated uterine cervical cancer is better in the morning than the afternoon. Nevertheless, the side effects of morning radiation do not differ significantly compared to the afternoon. The influence of the G₂-M phase proportion on the clinical response to radiation cannot be ascertained. The level of melatonin in the morning might affect the radiation response and affect the side effects on skin.

Keywords: cell cycle, cervical cancer, circadian, melatonin, radiosensitivity.

Abstrak :
The synergic effects of n-butanolic fraction of secondary metabolite of endophytic fungus 1.3.11 (FB) and doxorubicin (Dox) on cell cycle regulation and the expression of Bell - 2 gene expression were inventigated on MCF - 7 and T47-D cells by flow cytometry and immunocytochemical techniques respictively . The result showed that after 12 hours of incubation period with FB at its IC 50 dose, MCF-7 cell cycle was inhibited at G 1 phase while Dox inhibited the cell cycle at G2/M phase. Similar results were observed in T47 - D cells when incubated with DOX and FB individually under the same treatment condition. Further treatment was then performed to these cells where both DOX and FB were combined at their IC50 and 1/2 IC 50 dose and added to incubate with the cells over 12 hours period. Interestingly. the modified treatment combination showed that MCF - 7 D cell cycle regulation were inhibeted at G2/M phase. Our immunocytochemicalstudy also showed no significant inhibition suppresion of Bcl - gene expression in both MCF - 7 and T47 - D cells when compared with their corresponding positive control after treatment with FB and Dox or both combined FB and Dox at IC 30 and 1/2 IC 50 dosage over 15 hours incubation.