The Indonesian Biomedical Journal

350-4

Authors: Pudji Rahaju, Ayunita Tri Wirattami, Ferry Sandra, Steffi Kurniawan, Khairun Nisa, Soehartono Soehartono, Edi Handoko, Ahmad Dian Wahyudiono, Hendradi Surjotomo, Hendy Setyo Yudhanto, Nanik Setjowati

BACKGROUND: Progression of laryngeal carcinoma can be classified with the clinical staging, however there are different patterns of progressions observed in the patient with the same clinical stage which also affects their prognoses. Therefore biomarkers should be used. Nuclear factor (NF)-ĸB, Cyclin-D1, vascular endothelial growth factor (VEGF), cyclooxygenase (Cox)-2 have been reported for laryngeal carcinoma. However, it is still unclear how these markers are expressed and correlated in advanced stage laryngeal carcinoma. Therefore current study was conducted to investigate the expressions of NF-ĸB, Cyclin-D1, VEGF and Cox-2 and their correlations in advanced stage laryngeal carcinoma. METHODS: Subjects were recruited and laryngeal biopsies were collected, fixed in formalin and prepared for immunohistochemistry. The immunohistochemistry was performed using mouse monoclonal anti-NF-kB p65, anti-Cyclin-D12 anti-VEGF, and anti-Cox-2 antibodies. The immunohistochemistry results were documented and measured using ImmunoRatio. Pearson or Spearman correlation test was used based on the results of Shapiro-Wilk test of normality. A p-value of less than 0.05 is considered statistically significant. RESULTS: Twelve male subjects were included in this study. Expressions of NF-ĸB, Cyclin-D1, VEGF dan Cox-2 were clearly observed. Mean of NF-ĸB, Cyclin-D1, VEGF dan Cox-2 IHC expression levels measured with ImmunoRatio were 57.50±20.06%, 45.00±24.31%, 43.33±17.23% and 40.42±16.98%, respectively. There was significant correlation between the expressions of VEGF dan Cox-2 (p=0.031, r=0.622). CONCLUSION: Since correlation between the VEGF and Cox-2 expressions was statistically significant, VEGF and Cox-2 might have important roles in the growth, invasion and metastasis of laryngeal carcinoma.

364-74

Authors: Nur Ainina Abdollah, Nabil Izzatie Mohamad Safiai, Muhammad Khairi Ahmad, Kumitaa Theva Das, Siti Razila Abdul Razak

BACKGROUND: Molecular alterations of microRNA130a (miR130a) are observed in many types of cancers, including non-small cell lung cancer (NSCLC). However, the role of miR130a in NSCLC has been poorly studied. METHODS: In this study, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 was utilised to knockdown miR130a. The gRNA was designed to target the stem loop, 3’ and 5’ sites of miR130a and stably expressed in A549 cells. Post-treatment, mature levels of miR130a-3p and 5p were quantified, and proliferation and migration assays were conducted. RESULTS: Result showed significant suppression of miR130a-3p and -5p by two and three-fold respectively, when the CRISPR/Cas9 targeted at the 3’ site and stem loop of the miR130a gene. Suppression of miR130a-3p significantly increased the growth and migration of A549 cells, but no significant changes were observed in cells with suppressed expression of miR130a-5p. CONCLUSION: Our encouraging results highlight that the suppression of the miR130a is achievable using CRISPR/Cas9, and suppression of the miR-130a-3p could play an important role in the regulation of NSCLC.

383-95

Authors: Nurul Syuhada Nordin, Yong Yoke Keong, Siti Farah Md Tohid, Zetty Nadia Md Zain, Muhammad Nazrul Hakim

BACKGROUND: Eventhough 6-Mercaptopurine act as a major drug for rheumatoid arthritis (RA) treatment, however, its toxicity become a limitation. Therefore, this current study investigated whether 6-hydroxy-2-mercaptopurine (6H2MP) and 6-thioguanine (6TG) compounds are purine nucleoside analogues as a potential treatment of RA. The objective was to evaluate the therapeutics effects, especially the anti-inflammatory potential of 6H2MP and 6TG in the lipopolysaccharides(LPS)-induced RAW264.7 cells and phorbol myristate acetate (PMA)-induced HIG-82 cells. METHODS: Macrophage cells (RAW264.7) and rabbit synoviocytes (HIG-82) cells were induced using LPS and PMA to evaluate the anti-inflammatory potential of 6H2MP and 6TG. The cytotoxicity assessment was done by using MTT assay, while enzyme-linked immunosorbent assay (ELISA) was used to determine the anti‑inflammatory potential, including tumour necrosis factor (TNF-α), interleukins (IL-1β, and IL-6). RESULTS: Upon LPS-induced, RAW 264.7 macrophages demonstrated that 6H2MP and 6TG could suppress the production of nitric oxide (NO) in vitro. The half-maximal inhibitory concentration (IC50) value of 6TG and 6H2MP were 10.73 and 13.31, respectively. Further studied in PMA-induced HIG-82 synovial fibroblast cells showed that 6H2MP and 6TG also suppressed the release of NO, Prostaglandin E2 (PGE2), and inflammatory cytokines such as TNF-α, IL-1β and IL-6. The 6TG is more effective to reduce inflammatory reactions compared to 6H2MP, by the lower dose needed compared to 6H2MP in all experiments except in PGE2. CONCLUSION: The inhibition of inflammatory mediators is an important mechanism by which 6TG and 6H2MP may alleviate pain and articular inflammation. These results indicated that 6H2MP and 6TG are effective candidates for ameliorating inflammatory-associated complications.

403-8

Authors: Hamsu Kadriyan, Eka Sunarwidhi Prasedya, Nova Audrey Luetta Pieter, Masyita Gaffar, Amsyar Akil, Agusalim Bukhari, Budu Budu, Andi Alfian Zainuddin, Rina Masadah, Achmad Chusnu Rhomdoni, Abdul Qadar Punagi

BACKGROUND: Nasopharyngeal cancer (NPC) is known to release a specific exosome. NPC-derived exosome (NPC-Exo) could carry p53. However, information regarding the type of p53 carrier on NPC-Exo remains limited. This study aims to introduce our important findings regarding the type of p53 NPC-Exo cargo. METHODS: Serum from patients with NPC were prepared for exosome isolation with Seramir Exoquick by following the manual instructions. RT-PCR was conducted to determine the expression levels of latent membrane protein 1 (LMP-1) and p53 in the exosome isolate. Partial sequencing of p53 amplicon was conducted to determine mutation type of p53. RESULTS: There were 8 patients enrolled in this study. According to RT-PCR results, the expression levels of LMP-1 and p53 varied in the NPC-Exo isolate. Based on sequencing analysis, 1 case of p53 mutation was noticeable. CONCLUSION: According to current results, the NPC-derived exosome potentially carries not only wild type but also mutant type p53. Further research is needed to explore deeper the effect of the mutant type p53 as an exosome carrier in the clinical application.

418-25

Authors: Dina Keumala Sari, Ridha Dharmajaya, Mutiara Indah Sari, Dewi Masyithah Darlan

BACKGROUND: The presence of vitamin D receptor (VDR) polymorphism and high levels of inflammatory markers are predisposing factors indicating disease progression and malnutrition. To meet nutritional needs in maintaining nutritional status in tuberculosis patients with VDR gene polymorphisms (TaqI or FokI), food that is easily absorbed and high in vitamin D, calcium, and protein is needed. This study was conducted to determine whether high vitamin D and calcium porridge called soy–catfish–anchovy–rice (SCAR) porridge would increase 25(OH)D serum levels and other parameters, including calcium, albumin, high-sensivity C-reactive protein (hs-CRP), and blood glucose serum levels. METHODS: The study was a parallel, open, clinical trial. There were 22 subjects in the intervention (I) group who received 50 g of SCAR porridge once per day along with dietary counseling, and 21 subjects in the control (C) group who only received dietary counseling. All subjects with TC and CC (TaqI) or TC and CC (FokI) genotype were included in this study. The intervention lasted for 14 days, and the parameters such as 25(OH)D, calcium, albumin, hs-CRP, and blood glucose serum levels were assessed before and after intervention between groups. RESULTS: All subjects in both groups completed the study. After 14 days of intervention, there was a significant increase in 25(OH)D (p=0.01) and decrease in hs-CRP (p=0.02) serum levels in the I group, and no change was observed in the C group. There was no significant difference in albumin and blood glucose serum levels. CONCLUSION: The results show that 50 g of SCAR porridge per day for 14 days is an effective supplementation that can increase 25(OH)D and decrease hs-CRP serum levels in tuberculosis patients with VDR gene polymorphism.

433-7

Authors: Ferry Sandra, Jennifer Putri, Hilary Limen, Blanca Sarizta

BACKGROUND: The potential of the caffeic acid in other important Receptor Activator Nuclear Factor kB Ligand (RANKL)-Tumor Necrosis Factor (TNF)a-induced osteoclastogenic signaling pathways has not been known. Therefore, the current study was conducted to explore as well as to understand the inhibition potential of caffeic acid. METHODS: RAW264.7 cells were cultured, treated with caffeic acid, RANKL and TNFa. Tartrate Resistant Acid Phosphatase (TRAP) staining was performed to detect TRAP+ osteoclast-like polynuclear cells. To detect the activity of p44/42 Mitogen Activated Protein Kinase (MAPK), Akt, and Transforming Growth Factor-β-activated Kinase (TAK)1, the phosphorylated forms of the proteins were investigated with the immunoblotting assay. RESULTS: Pre-treatment of caffeic acid inhibited the RANKL and TNFa-induced differentiation of RAW264.7 cells into TRAP+ osteoclast-like polynuclear cells. RANKL and TNFa induced phosphorylation of p44/42 MAPK at Thr202/Tyr204, phosphorylation of Akt at both Ser473 and Thr308 and phosphorylation of TAK1 at Ser412. Pretreatment with caffeic acid prior to the RANKL and TNFa induction, inhibited the phosphorylation of MAPK, and TAK1, but not Akt. CONCLUSION: Caffeic acid might regulate the RANKL-TNFa-induced osteoclastogenic pathway in RAW264.7 by targeting TAK1, which later activation of p44/42 MAPK was abolished.