The Indonesian Biomedical Journal

124-31

Authors: Emilia Vivi Arsita, Dwi Aris Agung Nugrahaningsih, Ahmad Hamim Sadewa

BACKGROUND: Glutathione S-transferase Mu-1 (GSTM1) is known to undergo polymorphism and plays role in drug metabolism including Paclitaxel (PTX), the first-line chemotherapy for breast cancer. However, the effect of GSTM1 polymorphism against chemotherapy in breast cancer is limited and unexplored. This study was conducted to explore the effects of single and double guide (gRNA) on the GSTM1 knocked out (KO) and its effect on the response of PTX in the 4T1 cell line. METHODS: The preparatory stage was done by culturing and electroporating 4T1 cells using Ribonucleoprotein of clustered regularly interspaced short palindromic repeats (CRISPR)/Caspase 9 (Cas9). KO validation was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Sanger sequencing, and ICE analysis. The 4T1 viability was examined by MTT Assay. RESULTS: The number of base pairs of GSTM1 after being engineered by single or double gRNA was 86 bases. The DNA quantity of GSTM1 engineered by gRNA was more than using double gRNAs. The mRNA expression of GSTM1 engineered by single gRNA was lower than using double gRNAs. IC50 values of PTX between wildtype and KO were not significantly different, in the range of 30 µM. CONCLUSION: The base-pair length of GSTM1 exon 4 that is knocked out with single and double gRNA have the same number of base pairs. The quantity of GSTM1 DNA and mRNA expression are contrary between single gRNA and double gRNA, and IC50 PTX values in the 4T1 cell line of the control group with single or double gRNA knocked out do not differ markedly. PTX efficiency as chemotherapy is not disturbed in the GSTM1 deletion genetic profile.

141-9

Authors: Moordiani Moordiani, Dhania Novitasari, Ratna Asmah Susidarti, Muthi' Ikawati, Jun-ya Kato, Edy Meiyanto

BACKGROUND: Liver cancer is the third leading mortality in cancer. Curcumin shows effective anticancer potency against various cancer including liver cancer. The synthesized curcumin analog compounds Pentagamavunone-1 (PGV-1) and Chemoprevention Curcumin Analog-1.1 (CCA-1.1) have been well studied in breast, leukemia, and colon cancer cells with better potency than curcumin itself, yet their cytotoxic activities were not known in liver cancer cells. Thus, this study was conducted to elevate the anticancer effect of these curcumin analogs against hepatocellular carcinoma (HCC) cells in vitro, specifically in MYCN-expressing cells, based on its cellular physiology. METHODS: JHH-7 cells were used as the HCC cell model with high expression of MYCN. The viability of the cells was observed using trypan blue exclusion method while cell cycle profile and intracellular reactive oxygen species (ROS) levels were quantified by means of flow cytometry. Chromosomal staining with Hoechst was applied to determine the cell cycle arrest phase, whilst X-gal staining was used to assess the cellular senescence activity. RESULTS: The result of current study presented that the growth inhibitory activity of PGV-1 as well as CCA-1.1 in JHH-7 cells was associated with the cell cycle arrest and cellular senescence. Both curcumin analogs PGV-1 and CCA-1.1 ultimately induced mitotic arrest (p<0.001) better than curcumin. Moreover, PGV-1 and CCA-1.1 similarly increased the senescent cells that partly mediated through ROS elevation. The transcription level of MYCN was not altered upon treatment with curcumin and its analogs in JHH-7 cells, suggesting that molecular mechanism of the inhibitory effect was independent from MYCN signaling. CONCLUSION: Taken together, these observations revealed that both PGV-1 and CCA-1.1 potentially serve as multi-targeted curcumin-based compounds and lead to promising anti-hepatocellular cancer agents.

157-64

Authors: Ni Putu Sukma Sumantri Prabandari, Ni Kadek Mulyantari, Made Sukmawati, Anak Agung Ngurah Subawa, I Wayan Gede Artawan Eka Putra, Anak Agung Wiradewi Lestari, I Nyoman Wande, Ni Nyoman Mahartini

BACKGROUND: Early-onset neonatal sepsis (EONS) is an acute infection and sepsis occurring within the first 24 hours of new-born life. An increase in procalcitonin levels, presepsin levels, and neutrophil-to-lymphocyte ratio (NLR) in EONS subjects have been reported. However, whether presepsin levels and NLR affect the procalcitonin levels in EONS patients who have received antibiotic therapy has not been certainly known. This study was conducted to determine the correlation between presepsin levels and NLR with procalcitonin levels in EONS subjects. METHODS: A cross-sectional study involving 52 EONS subjects were conducted, and blood samples from subjects were collected. Presepsin levels were examined by enzyme-linked immunosorbent assay (ELISA) method, NLR was calculated from the absolute number of neutrophils divided by the absolute number of lymphocytes, and procalcitonin levels were examined by chemiluminescent microparticle immunoassay (CMIA) method. RESULTS: Median of procalcitonin levels and presepsin levels were 0.435 (0.12-9.11) ng/mL and 108.33 (71.43-1287.76) ng/L, respectively. While NLR value was 1.68 (0.2-7.52). There was significant difference between procalcitonin and presepsin levels (p=0.000), and so does between procalcitonin levels and NLR (p=0.001). Based on the multivariate analysis, presepsin levels also affected the procalcitonin levels (p=0.000; 95% CI: 0.001-0.004). CONCLUSION: The results of the study showed that there was significant correlation between presepsin levels and NLR with procalcitonin levels in EONS patients, suggesting that presepsin levels, NLR, and procalcitonin levels are potential candidates for EONS biomarkers.

171-8

Authors: Sonar Soni Panigoro, Sinta Chaira Maulanisa, Ahmad Kurnia, Denni Joko Purwanto, Primariadewi Rustamadji, Herqutanto Herqutanto, Ferry Sandra

BACKGROUND: Chemotherapy has reported to stimulate immune system through direct activation of cluster of differentiation (CD)8+ T cells. Neoadjuvant chemotherapy (NAC) is known to improve the clinical response of locally advanced breast cancer (LABC) patients. However, the immune response-related factor evaluation of NAC in LABC patients has not been routinely performed. Therefore, current study was conducted to evaluate the correlation of NAC-induced CD8+ T cell with chemotherapy response based on Miller Payne grading and World Health Organization (WHO) criteria. METHODS: LABC patients were recruited and data regarding age, gender, tumor, nodal stages, histopathological grade, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki67 were obtained. Biopsy and mastectomy tissues were collected and processed for hematoxylin-eosin and CD8 immunohistochemical staining. CD8+ T cell expression in peritumoral and intratumoral areas were documented and measured. Clinical responses based on Miller Payne grading and WHO were analyzed and correlated with CD8+ T cell expression. RESULTS: There were more subjects with high expression of total (80%), intratumoral (82.5%) and peritumoral (65%) CD8+ T cell expressions. The total (p=0.013) and intratumoral (p=0.015) CD8+ T cell expression, but not peritumoral CD8+ T cell expression, were significantly correlated with Miller Payne Grading. The total (p=0.009) and intratumoral (p=0.001) CD8+ T cell expressions were also significantly correlated with WHO clinical response. CONCLUSION: Total and intratumoral CD8+ T cell expressions are correlated with Miller Payne grading and WHO clinical response of NAC. Therefore, total and intratumoral CD8+ T cell expressions could be suggested as a predictive marker for clinical response of NAC.

187-93

Authors: Syanindita Puspa Wardhani, Khilmi Ainun Nadliroh, Al Mazida Fauzil Aishaqeena, Fahriza Abid Sonia, Achmad Guntur Hermawan Suryo Adji, Intan Masyfufah Hanim, Rizkia Milladina Hidayatulloh, Anna Fuji Rahimah, Ardian Rizal, Peter Sugita, Ferry Sandra, Djanggan Sargowo

BACKGROUND: Cardiometabolic syndrome is a risk factor for the development of diseases related to cardiovascular disease and decreased renal function. Ganoderma lucidum polysaccharide peptide (GLPP) has been reported to have anti-inflammatory and antioxidant properties. The current study was conducted to investigate the role of GLPP in inflammatory, oxidative stress and renal function markers of cardiometabolic subjects. METHODS: A randomized double-blinded perspective control trial with pre-post design was conducted. Cardiometabolic syndrome subjects were treated with placebo or GLPP for 60 days. Blood serum was collected from each subject before the first capsule consumption and one day after the last capsule consumption. Serum tumor necrosis factor (TNF)-α, high-sensitivity-C-Reactive Protein (hs-CRP) and malondialdehyde (MDA) levels were measured using enzyme-linked immunosorbent assay, while superoxide dismutase (SOD) level was measured using colorimetric assay. Serum urea and creatinine levels were measured using a clinical analyzer. The Cockroft-Gault formula was used to calculate estimated glomerular filtration rate (eGFR). RESULTS: Compared with the control group, the MDA level was significantly reduced, while the SOD level was significantly increased in the GLPP treatment group. Furthermore, serum urea and creatinine were lowered, while eGFR was increased in the GLPP treatment group. CONCLUSION: Treatment of GLPP for 60 days could be beneficial for lowering oxidative stress and improving renal function of patients with cardiometabolic syndrome.

25-46

Authors: Ferry Sandra, Andri Sutanto, Widya Wulandari, Reynaldo Lambertus, Maria Celinna, Nurrani Mustika Dewi, Solachuddin Jauhari Arief Ichwan

BACKGROUND: Pulp damage can lead to dentinogenesis impairment, irreversible pulpitis, or pulp necrosis. Despite being the most used endodontic procedure to treat damaged pulp, root canal therapy only results in nonvital teeth which are prone to fractures and secondary infection. Pulp-dentin regeneration has a potential to regenerate structure similar to normal pulp-dentin complex, and can be achieved by combining dental stem cells, scaffold, and signaling molecules. This article reviews the role of various types of dental stem cells, scaffolds, signaling molecules, and their combinations in regenerating pulp-dentin complex. CONTENT: Dental pulp stem cell (DPSC), stem cell from human exfoliated deciduous teeth (SHED), and dental follicle stem cell (DFSC) were reported to regenerate pulp-dentin complex in situ. SHED might be more promising than DPSCs and DFSCs for regenerating pulp-dentin complex, since SHED have a higher proliferation potential and higher expression levels of signaling molecules. Scaffolds have characteristics resembling extracellular matrix, thus providing a suitable microenvironment for transplanted dental stem cells. To accelerate the regeneration process, exogenous signaling molecules are often delivered together with dental stem cells. Scaffolds and signaling molecules have different regenerative potential, including induction of cell proliferation and migration, formation of pulp- and/or dentin-like tissue, as well as angiogenesis and neurogenesis promotion. SUMMARY: Combinations of dental stem cells, scaffold, and signaling molecules are important to achieve the functional pulp-dentin complex formation. Current trends and future directions on regenerative endodontics should be explored. The right combination of dental stem cells, scaffold, and signaling molecules could be determined based on the patients’ characteristics. Incomplete pulp-dentin regeneration could be overcome by applying dental stem cells, scaffold, and/or signaling molecules in multiple visits.

54-60

Authors: Soetjipto Soetjipto, Indri Dwi Murbani, Titut Harnanik

BACKGROUND: Lupus nephritis (LN) is associated with chronic renal failure and a high mortality rate. Tumor growth factor-β (TGF-β) plays a basic part in keeping up immune homeostasis, meanwhile extensive oxidation of the lipid membrane in LN causes the arrangement of malondialdehyde (MDA). Up to date, the mechanism of hyperbaric oxygen (HBO) therapy in LN has not been elucidated well. Hence this study was conducted to assess the impact of HBO therapy on TGF-β and MDA expression in the pristane-induced LN mice model. METHODS: Thirty-two mice aged 8-12 old weeks were isolated into 4 groups: normal saline-injected mice (group 1); pristane-injected mice (group 2); as well as pristane-injected followed by HBO-exposured with 2.4 (group 3) and 1.5 (group 4) atmosphere absolute (ATA) pressure. Pristane were injected intraperitoneal to initiate LN. Two months after pristane injection, the mice were placed within 24 hours in the special cage for metabolic examination to determined that pristane-induced LN mice model was successfully formed and marked by the occurring proteinuria. Oxidative stress was assessed by examining MDA, while systemic inflammation was assessed by examining by TGF-β. MDA and TGF-β serum were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: A significantly lower (p<0.050) MDA expression was found in group 3 in comparison with group 2. The results also showed that TGF-β level was significantly increased (p<0.050) in group 3 compared to group 2. CONCLUSION: HBO therapy ameliorates inflammation in LN by diminishing MDA and expanding TGF-β levels through activating antioxidants.

69-76

Authors: Vidia Sari, Riris Istighfari Jenie, Shofwal Widad, Agung Dewanto

BACKGROUND: The invasion of endometrial tissue in the peritoneal layers is an important precursor to the formation of endometriosis. However, the mechanisms of the initial development of endometrioma remain unclear. This study aimed to explore the correlation between matrix metalloproteinase (MMP)-9 activity and tissue inhibitors of metalloproteinase (TIMP)-1 expression in the initial development of endometriosis through the invasion process of endometrial lesions in extracellular matrix (ECM) remodeling. METHODS: A total of 30 samples of endometrioma tissue were obtained from 24 women with endometriosis and examined at Dr. Sardjito General Hospital, Yogyakarta, Indonesia. The tissue was then implanted into a chicken chorioallantoic membrane (CAM) for five days to examine its invasion ability. The invasion scores were assessed using hematoxylin-eosin staining. MMP-9 activity and TIMP-1 expression were analyzed using gelatin zymography and western blot, respectively. The correlations between MMP-9 activity and TIMP-1 expressions with the invasion scores were analyzed using Spearman correlation tests with significance set as p<0.05. RESULTS: The results showed that there was a strong positive correlation between MMP-9 activity and invasion scores of endometriomas tissue (r=0.656; p=0.000). Meanwhile, expression levels of TIMP-1 had a weak negative correlation with the increase of invasion scores of endometrioma (r=-0.388; p=0.034). These results indicate the involvement of MMP-9 and TIMP-1 in the initial development of endometriosis during the ECM remodeling. CONCLUSION: MMP-9 activity and TIMP-1 expression were correlated with the invasion score of endometrioma tissue. This study provides evidence for understanding the mechanisms involved in ECM remodeling in the early process of endometriosis.

85-93

Authors: Erick Yuane, Agung Dewanto, Shofwal Widad

BACKGROUND: Progestin therapy has been commonly used in endometriosis. The regulation of progesterone receptors B (PR-B) greatly affects the success rate of therapy in cases of endometriosis. The presence of tumor necrosis factor (TNF)-a in endometriosis triggers PR-B hypermethylation, decreasing PR-B expression and PR-B/A ratio that induce progesterone resistance. It may also occur in endometrioma. Studies regarding the distribution of PR-A and PR-B with TNF-a expression in endometriosis with endometrioma tissue samples has not been elucidated well. Therefore, this study was conducted to measure and compare the distribution of PR-A and PR-B expression, and to assess the effect of PR-B/A ratio on TNF-a in endometrioma and benign cysts. METHODS: A cross-sectional study was conducted by collecting paraffin blocks of endometriomas and benign cysts as controls, from patients undergoing surgery at Dr. Sardjito Hospital, Yogyakarta. Immunohistochemistry was performed to assess the expressions of PR-A, PR-B, TNF-a and PR-B/A ratio, to compared differences between endometriomas and benign cysts. RESULTS: Twenty-three endometrioma and 22 benign cyst tissue samples were collected. The mean PR-B expression and PR-B/A ratio were found to be lower in endometriomas than benign cysts, and mean expression of PR-A and TNF-a in endometriomas was higher than in benign cysts. However, there were no significant correlations between the expression of PR-A, PR-B, PR-B/A ratio, and TNF-a with endometriosis severity. CONCLUSION: In endometrioma cases, the expression of PR-A and TNF-a was higher, while the expression of PR-B and PR-B/A ratio was lower. However, there was no significant relationship between the ratio of PR-B/A and TNF-a.

100-5

Authors: Dewi Ranggaini, Ferry Sandra, Johni Halim, Solachuddin Jauhari Arief Ichwan, Melanie Sadono Djamil

BACKGROUND: Curcuma xanthorrhiza rhizomes have been demonstrated to have anticancer properties toward various types of cancer cells. The effect of C. xanthorrhiza rhizome extract (CXRE) on nasopharyngeal cancer (NPC) cells, including HONE-1 cell line has not been elucidated yet. Therefore, the effect of CXRE on the apoptosis of HONE-1 cells and its possible underlying mechanism are necessary to be explored. METHODS: C. xanthorrhiza rhizomes were minced, dried, extracted with distilled ethanol, filtered, and evaporated to produce CXRE. HONE-1 cells were seeded, starved, and treated with dimethyl sulfoxide (DMSO), Doxorubicin, or various concentrations of CXRE. Treated HONE-1 cells were stained with 4',6'-diamidino-2-phenylindole (DAPI) and the number of viable cells was counted. HONE-1 cells were also collected, lysed, and further processed for immunoblotting analysis to measure Bid activity. RESULTS: The number of viable HONE-1 cells decreased in concentration- and time-dependent manner. The number of viable cells in 50 and 250 μg/mL CXRE-treated groups were significantly lower compared with that in the DMSO-treated group after 24 h. At 48 h incubation period, the number of viable cells in 10, 50 and 250 μg/mL CXRE-treated groups were significantly lower compared with that in the DMSO-treated group. The number of viable cells in 250 μg/mL CXRE-treatment group was not significantly different compared with that in the Doxorubicin-treated group after 48 h. Bid expression levels in CXRE-treated groups were lower compared with that in the DMSO-treated group. CONCLUSION: CXRE could induce apoptosis via Bid activation, hence reducing the viability of HONE-1 cells.

349-57

Authors: Marwa Magdy Hamido, Nashwah Ismail Zaki, Sawsan Ahmed Nasr, Wael Mohamed El-Sayed

BACKGROUND: The interaction of carbamazepine (CBZ) and acetaminophen (APAP) could result in hepatic failure and mortality. This study was conducted to analyzed the potential of rosemary ethanol extract (REE) or coenzyme Q10 (CoQ10) to alleviate the interactions between CBZ and APAP. METHODS: Fourty-eight adult male rats were treated differently based on the assigned groups. Oxidative stress parameters, including malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase, and glutathione S-transferase (GST), and the expression levels of CYP3A4, CYP2E1, IL-6, TNF-α, and IL-1B in the liver were estimated. In addition, the histopathology of liver was examined and the plasma clearance rate of CBZ and APAP was estimated. RESULTS: Combination of CBZ and APAP significantly elevated alanine aminotransferase (ALT) activity and hepatic MDA, and reduced the activities of GPx, GST, and GSH level in liver. The gene expression of CYP3A4 and CYP2E1 was upregulated by CBZ and CoQ10, respectively. The expression of IL-6 has decreased in the groups treated with CBZ alone or in combination with APAP. TNF-α expression was significantly downregulated in the groups treated with CBZ, APAP, REE, CoQ10, or combination CBZ and APAP. The liver from CBZ and APAP combination group showed centrolobular degeneration and necrosis. REE and CoQ10 were able to alleviate most of these detrimental effects. The combined administration of CBZ and APAP extended the plasma clearance time of APAP and CBZ from 6 to 24 and from 9 to 24 hours, respectively. CONCLUSION: REE and CoQ10 alleviated the detrimental effects of the combination of CBZ and APAP through enhancing the cellular antioxidant milieu, induction of metabolizing enzymes, reduction of the plasma half-life of APAP and CBZ preventing their accumulation and potential interaction.

365-75

Authors: Sri Nabawiyati Nurul Makiyah, Masaki Kita, Ika Setyawati, Sri Tasminatun

BACKGROUND: Dioscorea alata L. tubers (DA) are suspected to prevent diabetes mellitus (DM) because they have a low glycemic index. However, only a few reports about the anti-diabetic effect of DA were reported up to date. This study aims to analyze the effect of DA consumption on several diabetic biomarkers through in vitro, in vivo, and in silico analysis. METHODS: In vitro experiments were conducted by observing the anti-inflammatory activity of DA extract, steroidal saponins (SDA) isolated from DA, and diosgenin in lymphocyte cell cultures. The tumor necrosis factor (TNF)-α and interferon (IFN)-γ percentages were analyzed by flow cytometry. In vivo study involved 24 healthy adolescents that were given a boiled DA 10 hours post-prandial. The blood sugar levels were measured at 0, 30, 60, and 120 min after treatment. Furthermore, the in silico study was carried out by analyzing the active compounds and predicting the biological activity, the target proteins, and interactions of target proteins with compounds contained in DA . RESULTS: DA extract, SDA isolated from DA, and diosgenin at 50 µg/mL significantly reduced pro-inflammatory cytokines TNF-α and IFN-γ in lymphocyte cell culture. The blood glucose levels in the DA group were lower at 30 and 60 min after treatment. Based on the in silico study, the anti-diabetic activity of DA was speculated to be attributed to the mechanisms of anti-hyperglycemia, prevention of mitochondrial dysfunction, anti-inflammation, and treated insulin resistance. Several proteins included in the DM pathway became the protein target of compounds contained in DA. CONCLUSION: DA potentially have an anti-diabetic activity through several mechanisms.

382-92

Authors: Soraya Rahmanisa, Ekavianty Prajatelistia, Indra Wibowo, Anggraini Barlian

BACKGROUND: Biosilica derived from Indonesian marine sponge Melophlus sarasinorum and Xestospongia testudinaria is one of the biomaterials that can be developed together with synthetic polymer as a composite. Poly E-caprolactone (PCL) used as a composite role as an osteoconductive material together with biosilica and also tailored the slow rate of degradation in the body. This study aimed to create a biocompatible biosilica-based scaffold and supports osteogenic differentiation of human Wharton's Jelly mesenchymal stem cell (hWJ-MSCs). METHODS: Biosilica was extracted from M. sarasinorum and X. testudinaria with the acid digestion method. Scaffold was prepared using the salt leaching method. The composite scaffolds were made from seven different biosilica extract and PCL. All of the scaffolds were tested for the cell morphology, Fourier-transform infrared spectroscopy (FTIR), immunocytochemistry, and cytotoxicity. RESULTS: Composite scaffolds of 50% M. sarasinorum and X. testudinaria increased the cell viability and supported the cell growth within 14 days, whereas the osteogenic differentiation can be seen by the presence of collagen type 1 in day 12 based on immunocytochemistry result. CONCLUSION: The biosilica scaffolds from PCL+50% M. sarasinorum and PCL+50% X. testudinaria were promising 3D scaffolds for potential application in bone tissue engineering. In conclusion, this study shows evidence for the osteogenic differentiation of hWJ-MSC, which might be developed for bone tissue engineering.

401-9

Authors: Banun Kusumawardani, Larissa Tania, Ari Satia Nugraha

BACKGROUND: Cervical cancer has a high rate of morbidity and mortality in women with cancer. Recent studies have found that tobacco (Nicotiana tabacum L.) is a potential source of anti-cancer agents. Hence, this study was conducted to determine the potential of Kasturi tobacco leaf resinoids as apoptotic agents against cervical cell malignancies, since it has not been fully elucidated before. METHODS: The phytochemical diversity of Kasturi tobacco resinoids was generated by gas chromatography-mass spectrometry (GC-MS) analysis followed by spectral similarity to National Institute of Standards and Technology (NIST) database. Cytotoxicity and proliferative activity of HeLa cells treated with Kasturi tobacco resinoids at various concentrations were evaluated by MTT assay. The expression of Caspase-3, cyclooxygenase-2 (COX-2) and heat shock protein 90 (HSP-90) in HeLa cells was analyzed by immunocytochemistry. Next, the migration ability of HeLa cells was observed by the scratch method. RESULTS: Kasturi tobacco resin contains 4,8,13-cyclotetradecatriene-1,3-diol, 1,5,9-trim with α-2,7,11-cembratriene-4,6-diol (α-CBD) structure in the form of a diterpenoid compound with the chemical formula C20H34O2 and a molecular weight of 306 Da. Kasturi tobacco resinoid with IC50 value of 2500 μg/mL inhibited proliferative activity during 72 hours. At a concentration of 1¼ IC50 and incubation for 48 hours, Caspase-3 expression increased by 74.1%, while COX-2 and HSP-90 expression decreased by 28.3% and 26.1%, respectively. HeLa cell migration was inhibited by Kasturi tobacco resinoid at 24 hours incubation. CONCLUSION: Kasturi tobacco resinoids with a concentration of 1¼ IC50 have potential as cervical anti-cancer agents by increasing Caspase-3 expression and decreasing COX-2 and HSP-90 expression within 48 hours.

416-20

Authors: Ferry Sandra, Dewi Ranggaini, Laifa Annisa Hendamin, Nurrani Mustika Dewi, Melanie Sadono Djamil

BACKGROUND: Formation of tumour mass is one symptom of osteosarcoma development. Caffeic acid has been known to provide effective treatment but has less side effect for some cancer therapy. Studies reported that caffeic acid might promote apoptosis in MG-63 osteosarcoma cells, however, the effect of caffeic acid treatment in preventing tumour mass formation has not been well elucidated, especially in MG-63 cells-induced nude mice in vivo. METHODS: MG-63 cells were pre-treated with 0, 1, or 10 µg/mL caffeic acid, and 6 hours after pre-treatment, MG-63 cells were injected into subcutaneous space of mice to induce osteosarcoma. Another model was also created by subcutaneously injecting MG-63 cells to the back of mice, and after 48 days, the visible tumour mass was injected intra-tumour with 0 or 10 µg/mL caffeic acid every 7 days for 6 times. After 90 days, mice were anaesthetised, and the nodule pictures were taken for observation and measurement. RESULTS: In pre-treated MG-63 cells-induced mice, volumes of the mass decreased in reverse with the dose of caffeic acid given. Ten µg/mL caffeic acid pre-treatment was able to significantly lower the mass volume compared to the untreated (p<0.05). Meanwhile, the intra-tumour treatment of 10 µg/mL caffeic acid, even though not significant, was able to inhibit tumour mass formation. CONCLUSION: Results of caffeic acid pre-treatment and caffeic acid treatment in tumour mass of mice show that caffeic acid is able to inhibit the MG-63 cells formation. This suggests that caffeic acid can be a potential anti-cancer agent.

429-35

Authors: Jonathan Alvin Nugraha Halim, Endang Sri Lestari, Sigit Adi Prasetyo, Muflihatul Muniroh, Agung Aji Prasetyo

BACKGROUND: Cholestasis caused by obstruction of the common bile duct and may developed gut-derived sepsis due to reactive oxygen species (ROS) accumulation. Ursodeoxycholic acid (UDCA) and glutathione are widely known for their antioxidant properties, that might be beneficial against ROS. However, the effects of UDCA-glutathione combination against ROS have not been well elucidated in previous studies. Thus, this study was conducted to evaluate tumor necrosis factor (TNF)-α level and height of terminal ileal mucosal villus after UDCA-glutathione administration in cholestasis rat model. METHODS: Twenty-eight male Sprague Dawley rats were randomly grouped into four treatment groups, each group consisted of seven rats that had previously undergone bile duct ligation. Three groups received treatment of UDCA-glutathione combination on stratified dose, while the other one only received UDCA. Each treatment was given for 21 days. Ileal samples were collected from the rats and stained with mouse anti TNF-a antibody and hematoxylin-eosin (HE). Immunohistochemistry and histopathological examination were done using microscope and then calculated with ImageJ. RESULTS: The combination of UDCA and glutathione treatment decreased the TNF-α expression (p<0.05) compared to UDCA only group, particularly in group that received 20 mg UDCA and 15 mg glutathione supplementation (p<0.05) and group that received 30 mg UDCA and 20 mg glutathione supplementation (p<0.05). The height of the mucosa villous was higher in the UDCA-glutathione combination groups for all the three dosage variations given (p<0.05) compared to UDCA only group. CONCLUSION: UDCA-glutathione combination downregulates TNF-α expression and improves ileum mucosal villus height in cholestasis.

243-51

Authors: Raghda Abdulsamad Abdualhay, Adnan Jassim Mohammed Al-Fartosy

BACKGROUND: Gestational diabetes mellitus (GDM) has a strong relationship with an increased risk of maternal and perinatal complications. However, in Basrah, Iraq, studies regarding GDM are still limited. In current study, we aimed to investigate the association between insulin resistance and some clinical predictors of GDM among pregnant women in 1st and 3rd trimesters of gestation. METHODS: This case-control study was conducted on 44 pregnant women with GDM and 45 without GDM aged 20 to 40 years who applied for GDM screening during the first (9-13 week) and third trimester (24-28 week) of pregnancy. Demographics, blood glucose, HbA1c, insulin, homeostatic model assessment for insulin resistance (HOMA-IR), spexin, nesfatin-1, orexin-A, vaspin and lipid profile levels were compared between groups. RESULTS: Subjects with GDM showed a higher level of glucose, insulin HOMA-IR, HbA1c, spexin, vaspin in the first and third trimesters of pregnancy (p<0.01) compared to the healthy subjects. Meanwhile in the first and third trimester, subjects with GDM showed significantly lower level of nesfatin-1 and orexin-A compare to the control. In third trimester, oral glucose tolerance test (OGTT) outcomes for fasting glucose at 1 hour, 2 hours, and 3 hours after glucose load were significantly higher (p<0.01). According to the area under the receiver operating characteristics (ROC) curve (AUC) findings, HOMA-IR, spexin, and vaspin may be more effective predictors biomarkers for GDM in pregnant subjects, while orexin-A and nesfatin-1 were ineffective. CONCLUSION: The correlation of insulin resistance and adipokines in the first and thrid trimester was not significantly different, which may cast new light on the possible role as an etiological cause of GDM and might be a better monitoring parameter in women with GDM.

261-8

Authors: Kelvin Supriami, Ira Puspitawati, Dyah Samti Mayasari, Anggoro Budi Hartopo

BACKGROUND: Platelet-derived microparticles (PDMPs) and low-density lipoprotein (LDL) cholesterol are contributing factors to acute myocardial infarction (AMI). However, the association between LDL cholesterol and PDMPs in AMI has not fully discovered. This study assessed the correlation between these two parameters in patients diagnosed with AMI. METHODS: This was an observational cross-sectional study involving 95 subjects with AMI. The blood measurement of PDMPs counts and LDL cholesterol levels were conducted concomitantly within 24 hours of admission. PDMPs count was analyzed by flow-cytometry method, meanwhile the LDL cholesterol was measured with enzymatic and colorimetric methods. For further analysis, subjects were further divided into LDL cholesterol level ≥130 mg/dL and <130 mg/dL. A statistical test was conducted for a correlative and comparative analyses. RESULTS: A correlative analysis to assess the association between PDMPs counts and LDL cholesterol level depicted a low but significant positive correlation (r=0.231, p=0.024). Furthermore, mean PDMPs counts was significantly higher in subjects with LDL cholesterol level ≥130 mg/dL compared to LDL cholesterol level <130 mg/dL (12,499.59 (95% CI: 8,507.44-16,491.74) counts/μL vs. 9,267.23 (95% CI: 4,445.45-14,089.01) counts/μL; p=0.039). CONCLUSION: There was a significant correlation between PDMPs counts and LDL cholesterol levels in AMI. A significantly increased PDMPs counts were found in subjects with LDL cholesterol level ≥130 mg/dL. Therefore, it is recommended to measure PDMPs in patients with high LDL cholesterol levels as both might be significant AMI biomarkers.

276-81

Authors: Ferry Sandra, Muhammad Ihsan Rizal, Nurrani Mustika Dewi, Toshio Kukita

BACKGROUND: Increase in inflammatory cytokine levels promotes pathological osteoclast differentiation. Caffeic acid has anti-inflammatory properties and can inhibit osteoclast bone resorption. In vitro studies have reported the ability of caffeic acid in inhibiting osteoclastogenesis pathways, however the in vivo study is rarely conducted. The aim of this study is to examine the role of caffeic acid in reducing inflammation and inhibiting osteoclastogenesis in Adjuvant-Induced Arthritis (AIA) rats. METHODS: Rats were injected with Freund’s Complete Adjuvant (CFA) and mineral oil. One day after injection, various concentration (0, 5, 25, 125 mg) of caffeic acid were given gastro-intestinally. Swelling degree in rats’ ankle joints was determined by measuring height and width of each ankle joint. Bone loss level was examined with soft X-ray, and then bone density was calculated. To examine osteoclastogenesis, ankle joints were stained with Tartrate-Resistant Acid Phosphatase (TRAP) and evaluated microscopically. RESULTS: Ankle joints of AIA rats had severe swelling before treated, yet the swelling was reduced based on concentration-dependent after receiving caffeic acid. Severe bone loss in AIA rats’ ankle joints were also observed, however the treatment of 125 mg caffeic acid showed remarkable inhibition effect toward rats’ bone loss. Osteoclastogenesis in AIA rats’ ankle joints were higher than the normal ones, as indicated with high TRAP-positive Multinucleated Cells (MNCs). But low number of TRAP-positive MNCs was observed in ankle joint of AIA rats that received 125 mg caffeic acid. CONCLUSION: Administration of caffeic acid can reduce the degree of swallowing, inhibit bone loss, and inhibit osteoclastogenesis in ankle joint of arthritis-induced rats.

289-93

Authors: I Ketut Edy Sudiarta, Monicha Zalzabilla Aldinasyah, Cindy Jennilyn Candra, Supriyono Supriyono, Annisa Ullya Rasyida

BACKGROUND: Premature Rupture of Membranes (PROM) is significantly linked to the infections-related maternal deaths. In the inflammatory process, the influencing stressor will stimulate the activation of Nuclear Factor Kappa B (NF-κB) and Receptor of Advanced Gyclation End product (RAGE). Yet up to date, the expression of NF-κB and RAGE in pregnant women with PROM are still rarely studied. Therefore, this study aimed to observe the differences of NF-κB and RAGE expression from PROM and non-PROM subjects. METHODS: This was a cross-sectional study involving 20 PROM subjects and 20 non-PROM subjects with infections and complications. Samples from the fetal membrane tissue of subjects were obtained and put into paraffin block preparation for the determination of NF-κB and RAGE expression. The detection of NF-κB and RAGE expression was conducted using immunohistochemical staining and observed under an upright light microscope. The expressions were later calculated using ImageJ software. RESULTS: Both NF-κB and RAGE expression were found to be higher in PROM subjects compare to the non-PROM subjects. The median of NF-κB in PROM and non-PROM subjects were 32.47±1.22 and 5.59±1.09, respectively (p=0.000). While the median of RAGE in PROM subjects was 53.58±3.46, and in non-PROM subjects was 11.64±2.49 (p=0.013). CONCLUSION: There is significant difference between NF-κB and RAGE expression in fetal membranes of PROM and non-PROM subjects. Therefore, the increased of NF-κB and RAGE expression can be used as a potential marker to detect complication of PROM.

303-8

Authors: Nataniel Tandirogang, Silvia Anitasari, Enos Tangke Arung, Swandari Paramita, Yung Kang Shen

BACKGROUND: Indonesia's tropical forest is home to around 80% of the world's medicinal plants. One of these is Zingiber purpureum, which have traditionally been used to treat joint discomfort, the common cold, and jaundice. The rhizomes of this plant have been suggested to possess antibacterial action in the treatment of infections. In this study, Z. purpureum was screened for antibacterial activity against 13 bacteria (Gram-positive and Gram-negative). METHODS: Z. purpureum rhizomes were obtained and the distillated extracts were made to generate essential oil. The minimum inhibitory concentration (MIC) and Kirby Bauer disk diffusion methods were used to determine the antibacterial activity. RESULTS: All bacteria activity were inhibited by the essential oil of Z. purpureum at concentrations ranging from 2.5 vol% to 10 vol%. However, several bacterias (Escherichia coli, Pseudomonas aeruginosa, and Enterobacter cloacae) were inhibited at the lowest concentration (0.63 vol %), with the inhibition zones ranging from 6.7 mm to 8.0 mm. Meanwhile, the widest inhibition zone (13.3 mm) was reported on E. cloacae at 10 vol% concentration. CONCLUSION: A 10 vol% Z. purpureum rhizome extract inhibits Gram-positive and Gram-negative bacteria, particularly those that are resistant to a variety of antibiotics.

316-22

Authors: Valentinus Besin, Abdul Hafid Bajamal, I Ketut Sudiana, Mangestuti Agil, Jusak Nugraha, Mohammad Hasan Machfoed, Paulus Budiono Notopuro, Naesilla Naesilla

BACKGROUND: Interleukin (IL)-10 is involved in Wallerian degeneration after peripheral nerve crush injury. Oral thymoquinone was previously observed to decrease local immunoglobulin-G (IgG) in a crush-injured rat model. No study has evaluated the pathway of various thymoquinone dosages on local IgG and IL-10 in this injury. METHODS: This experimental study used 126 Rattus norvegicus Wistar rats that were divided into 18 groups: six groups received a placebo, the other six groups received thymoquinone at 100 mg/kg/day and the last six groups received thymoquinone at 250 mg/kg/day, respectively. Rats were sacrificed at 12, 18, 24, 5x24, 6x24, and 7x24 hours. Matrix metalloproteinase-9 (MMP-9), IL-10, and local IgG levels were assessed by Enzyme-Linked Immunosorbent Assay (ELISA). The nuclear factor KappaB (NF-κB) expressions on Schwann cells were examined by flow cytometry. Path analysis was performed using SmartPLS. RESULTS: The path analysis showed that 100mg/kg/day of thymoquinone significantly decreased NF-κB expression. However, NF-κB did not affect local MMP-9, and MMP-9 had no significant relationship with local IL-10 and IgG. Thymoquinone 250 mg/kg/day also significantly inhibited NF-kB expression, decreased local MMP-9, and, in turn, decreased local IL-10 and IgG. CONCLUSION: Administration of oral thymoquinone 250 mg/kg/day decreases local IgG and IL-10 levels via suppressing NF-κB expression and MMP-9 levels.

139-47

Authors: Nor Raihan Mohammad Shabani, Che Muhammad Khairul Hisyam Ismail, Chiuan Herng Leow, Munirah Mokhtar, Kirnpal Kaur Banga Singh, Chiuan Yee Leow

BACKGROUND: Shigella is a Gram-negative rod-shaped intracellular bacterial pathogen that causes bacterial dysentery or shigellosis among children under five years old. Antibiotics have been less effective in treating shigellosis due to the multi-drug resistance of Shigella. Therefore, an effective vaccine is urgently needed to prevent this disease. The present study aims to determine the peptides presented by major histocompatibility complex (MHC) class II molecules of Shigella-infected macrophages using mass spectrometry-based immunopeptidomics approaches. The MHC class II-associated peptides derived from Shigella-infected macrophages are candidates for developing subunit-based Shigella vaccine. METHODS: THP-1-derived macrophages were infected with Shigella flexneri 2a at the multiplicity of infection equal to 10. The lysate was immunoprecipitated and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). The sequences retrieved were analyzed using bioinformatics tools. RESULTS: The Shigella-infected THP-1-derived macrophages contained sample peptides from source proteins of almost all subcellular localizations. Eight peptides from S. flexneri 2a-infected macrophages were predicted to be localized at the outer membrane proteins (OMPs) of S. flexneri 2a by the PSORTb server. Two of the OMP-associated peptides were predicted as antigenic, non-allergenic, and non-toxic by respective bioinformatics tools. CONCLUSION: The findings in this study showed two selected OMPs have great potential for vaccine development against shigellosis.

156-63

Authors: Hiqmah Yusi Yana, Lisna Hidayati, Nastiti Wijayanti, Tri Rini Nuringtyas

BACKGROUND: Aquilaria malaccensis has been consumed as herbal medicine, and in vitro study showed that the leaf extract possesses high antioxidant activities. A brief preliminary study indicated that A. malaccensis showed a promising immunomodulatory activity when evaluated using latex beads. This current study aimed to evaluate the immunomodulatory activity of A. malaccensis leaf extract on the macrophage, which was challenged with pathogenic bacteria Staphylococcus aureus. METHODS: Bioactivity was determined by evaluating the phagocytic capacity of macrophages isolated from Mus musculus against S. aureus. First, the cytotoxicity of extracts on macrophages was evaluated using MTT assays, and the IC50 value was used to determine the dose of immunomodulatory assays. The highest toxicity was observed on chloroform extract with an IC50 value of 111.4 µg/mL. Therefore, the treatment was 100 and 50 µg/mL. Two parameters, including the phagocytic activity and phagocytic capacity of macrophages infected with S. aureus, were used to evaluate immunomodulatory activity. The analysis of variance was done at p<0.05 to determine the significant difference among treatments. RESULTS: Chloroform and ethanol extracts at a 50 µg/mL concentration showed the best results with the phagocytic activity of 82.33%±9.61% and 80.33±1.53%. The ethyl acetate showed lower phagocytic activities of 70.67±1.53. All extracts significantly increased phagocytic activity and phagocytic capacity, and the results differed significantly between negative and positive controls. Thin-layer chromatography indicated that the extract contained terpenoid, flavonoid, phenolic, and tannin. CONCLUSION: A. malaccensis leaf extracts showed immunomodulatory activity. Both chloroform and ethanol extracts showed comparable activity, while the ethyl acetate extract was lower. The extracts contained diverse bioactive compounds that may support activating macrophage cells for immunomodulatory activity.

172-9

Authors: Ferdian Ramadhan, Jamaluddin Jamaluddin, Ismi Zuhria, Luki Indriaswati, Evelyn Komaratih

BACKGROUND: Pterygium is an ocular surface disease that often occurs in tropical countries with a high recurrence rate. Matrix metalloproteinase-3 (MMP-3) play a key role in the inflammatory process of pterygium. This study aims to investigate the ability of curcumin and fibrin glue (FG) in suppressing the expression of MMP-3, and whether can be expected as adjuvant therapy to reduce pterygium recurrence. METHODS: Human pterygium fibroblasts (HPF) obtained from primary cultured of pterygium were treated with no treatment, curcumin, mitomycin-C (MMC), and FG. MMP-3 expression was analyzed using immunocytochemistry and the intensity measurement was done using ImageJ software. Cell migration was measured by scratching and stratification of fibroblast culture after cell confluence, and assessed for 48 hours. RESULTS: The expression of MMP-3 were lower in the HPF treated with 100 mol/mL curcumin, 200 mol/mL, and FG (2205.84±86.1 pg/mL, 1002.51±25.22 pg/mL, 1131.55±17.71 pg/mL, respectively) in comparison with untreated HPF (4703.49±108.9 pg/mL). The expression of MMP-3 were significantly different between groups (p<0.001). Cell migration of HPF after scratching with curcumin intervention at 200 mol/mL decrease from 178.67±2.85 (24 hours) to 88.83±1.48 (48 hours). Meanwhile the migration in FG group also decrease from 180.4±2.56 (24 hours) to 72.45±1.25 (48 hours). CONCLUSION: Curcumin and FG able to reduce the expression of MMP-3 and inhibit the migration of HPF cells.

186-92

Authors: Erlina Sih Mahanani, Ikhsan Nur Arifin, Arya Nur Ihsan, Yusrina Lukitasari, Ferry Sandra

BACKGROUND: Parkia speciosa seeds have been reported to have an anticancer property due to the presence of various antioxidant compounds. Since the potential uses of P. speciosa for the tongue cancer has not been clearly disclosed, we conducted a study to investigate anticancer properties of P. speciosa seed ethanol extract (PSSEE) as well as its effect on cardiac cells. METHODS: Tongue cancer rat model were treated with/without Doxorubicin and various concentrations of PSSEE. After treatment, tongue and heart samples were collected and processed further for histological examinations. Tongue epithelium thickness and damaged heart tissues was observed by HE staining, while tongue cancer cell proliferation was assessed by Ki-67 immunohistochemistry. Analyses were performed under an upright light microscope to measure tongue epithelium thickness, state of cancer cell proliferation, and degree of heart tissue damage. RESULTS: Addition of 400 mg/kg body weight (BW) PSSEE to 4.6 mg/kg BW Doxorubicin reduced the average tongue epithelial thickness and Ki-67+ cells number. Upon addition of PSSEE to Doxorubicin, the damage of heart tissue was reduced in a concentration dependent manner. Among all groups, the group of tongue cancer treated with 4.6 mg/kg BW Doxorubicin and 400 mg/kg BW PSSEE had the lowest percentage as well as the lowest degree of heart tissue damage. CONCLUSION: Since addition of PSSEE to Doxorubicin reduced epithelial thickness, number of Ki-67+ cells and heart tissue damage, PSSEE could be a potential co-chemotherapeutic agent for Doxorubicin toward tongue cancer.

199-205

Authors: Bertoka Fajar Surya Perwira Negara, Jae Suk Choi

BACKGROUND: Bifidobacterium is a genus of lactic acid bacteria that lives in the large intestine of humans and animals. The health benefits of this genus are well established; however, the anti-inflammatory activity of this genus, specifically Bifidobacterium lactis, has not been well defined. Therefore, in this study, we evaluated anti-inflammatory activity of B. lactis hydrolysates using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. METHODS: RAW 264.7 cells were cultured using Dulbecco’s Modified Eagle’s Medium in 5 % CO2 incubator at 37 ℃. One µg/mL of LPS was used to stimulate RAW 264.7 cells. Nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were measured to evaluate anti-inflammatory activity of B. lactis hydrolysates. The cytotoxicity of the inhibitor was also measured in present study through 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. RESULTS: The results showed that B. lactis hydrolysates at 25–200 μg/mL inhibited NO production. In concentration-dependent manner, B. lactis hydrolysate showed inhibition of iNOS expression. However, no inhibition on COX-2 expression was observed. The MTS assay of the B. lactis hydrolysates showed no side effects on the cell viability at all concentrations. CONCLUSION: The current study revealed that B. lactis hydrolysates possess specific anti-inflammatory effects by inhibiting iNOS expression without cytotoxicity and therefore could potentially be developed as a new iNOS inhibitor.

211-7

Authors: Ni Ketut Susilawati, I Wayan Putu Sutirta Yasa, Wayan Suardana, Sri Maliawan, I Made Jawi, Achmad Chusnu Romdhoni, Hamsu Kadriyan, I Wayan Gede Artawan Eka Putra, I Gusti Kamasan Nyoman Arijana

BACKGROUND:Purple sweet potato tuber ethanol extract (PSPTEE) has been known to have benefits in various disease, including to prevent nasopharyngeal carcinoma (NPC). However, there has been no research on PSPTEE in preventing the proliferation of nasopharyngeal mucosal epithelial cells induced by formaldehyde through the oxidative stress mechanisms and inflammatory process. Studies have proven the effect of PSP on various cancer cells, but the carcinogenesis process of the nasopharyngeal mucosal epithelium is still limited. Hence, this study aimed to demonstrate the mechanism for preventing histopathology changes with PSPTEE due to formaldehyde exposure. METHODS: Thirty-two formaldehyde-induced Wistar rats were treated with or without 1g/kgBW/day PSPTEE for 16 weeks. Malondialdehyde (MDA) level were examined with spectrophotometry method, while tumor necrosis factor (TNF)-α and p53 with enzyme linked immunosorbent assay (ELISA) method. The histopathology appearance of the nasopharyngeal epithelium was observed with hematoxylin-eosin (HE) staining. RESULTS: MDA and TNF-α levels in control and treatment group were 22.89±2.84 μM; 9.83±0.89 μM and 84.18±11.58 ng/L; 73.92±10.59 ng/L, respectively, and they showed significant difference. Meanwhile, the level of p53 showed no significant difference. Histopathology appearance showed a significant difference. Path analysis for MDA, TNF-α and p53 levels contributed 28.7% to histopathology appearance. MDA have an effect on significant direct effect on TNF-α. TNF-α has a significant direct effect to histopathological appearance. Indirect influence MDA on histopatological appearance were smaller than the direct effect. TNF-α has the greatest influence on histopatological appearance. CONCLUSION: Formaldehyde induces the histopathology appearance in nasopharyngeal epithelium. This study also demonstrates the mechanism to prevent the nasopharynx epithelial histopathology appearance by administration of PSPTEE.