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Cyntia Gracesella Patintingan,1 Melva Louisa,2 Vetnizah Juniantito,3 Wawaimuli Arozal,2 Silmi Hanifah,1 Septelia Inawati Wanandi,4 Rajarajan Thandavarayan5 1Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; 2Department of Pharmacology and Therapeutics, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; 3Department of Veterinary Clinic Reproduction and Pathology, Faculty of Veterinary Medicine, Agriculture Institute of Bogor, Bogor, Indonesia; 4Department of Biochemistry and Molecular Biology, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia; 5Department of Cardiovascular Sciences Houston Methodist Research Institute, Houston, TX, USACorrespondence: Melva Louisa, Department of Pharmacology and Therapeutics Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia, Tel +62-21-31930481, Email melva.louisa@gmail.comBackground: Doxorubicin, an anthracycline class of anticancer, is an effective chemotherapeutic agent with serious adverse effects, mainly cardiotoxicity. Several possible causes of doxorubicin cardiotoxicity are increased oxidative stress, nucleic acid and protein synthesis inhibition, cardiomyocyte apoptosis, and mitochondrial biogenesis disruptions. Moringa oleifera (MO), a naturally derived medicine, is known for its antioxidative properties and activity in alleviating mitochondrial dysfunction. To determine the potency and possible cardioprotective mechanism of MO leaves aqueous extract via the mitochondrial biogenesis pathway in doxorubicin-induced rats.Methods: Twenty-four Sprague-Dawley rats were divided into four groups of six. The first group was normal rats; the second group was treated with doxorubicin 4 mg/kg BW intraperitoneally once weekly for four weeks; the third and fourth groups were treated with doxorubicin 4 mg/kg BW intraperitoneally once weekly, and MO leaves extract at 200 mg/kg BW or 400 mg/kg BW orally daily, for four weeks. At the end of the fourth week, blood and cardiac tissues were obtained and analyzed for cardiac biomarkers, mitochondrial DNA copy number, mRNA expressions of peroxisome-activated receptor-gamma coactivator-1 alpha (PGC-1α), the nuclear factor erythroid 2-related factor 2 (Nrf2), superoxide dismutase 2 (SOD2), caspase 3, the activity of glutathione peroxidase (GPx), levels of 8-hydroxy-2-deoxyguanosine (8-OH-dG), and malondialdehyde.Results: MO leaves extract was shown to decrease biomarkers of cardiac damage (LDH and CK-MB), malondialdehyde levels, and GPx activity. These changes align with the reduction of mRNA expressions of caspase-3, the increase of mRNA expressions of PGC-1α and Nrf2, and the elevation of mitochondrial DNA copy number. MO leaves extracts did not influence the mRNA expressions of superoxide dismutase 2 (SOD2) or the levels of 8-OH-dG.Conclusion: Moringa oleifera leaves extract ameliorates doxorubicin-induced cardiotoxicity by reducing apoptosis and restoring gene expression of PGC-1α and Nrf2, a key regulator in mitochondrial biogenesis.Keywords: anthracycline, DNA damage, mitochondrial DNA, Moringa oleifera Lam, oxidative stress

Objective: This cohort study aimed to examine the impact of the FLT3-ITD mutation on the downstream signaling pathway of PI3K/AKT pathway, the percentage of leukemia stem cells, and the survival of patients receiving D3A7 induction therapy., Method: Bone marrow mononuclear cells were collected from 20 adult AML patients who had completed D3A7 induction therapy at Cipto Mangunkusumo National General Hospital and Dharmais Cancer Hospital. FLT3-ITD gene mutation was examined by the PCR-sequencing method. Expression of phosphorylated PI3K and AKT was detected using the sandwich ELISA method. Flow cytometry was used for detecting the number of apoptosis and proliferation cells, and biomarkers of leukemia stem cells., Result: The expression levels of PI3K and AKT proteins were higher in FLT3-ITD, both in the mutant group compared to the non-mutation group, and in the patient group with treatment failure outcomes compared to the patient group with treatment response. The percentage of the leukemia stem cell population did not differ significantly between the FLT3-ITD mutation group and the wild type group, and between the treatment failure outcome group and the response outcome group., Conclusion: This study presents the important role of FLT3-ITD mutation via its downstream signaling (PI3K/AKT) in the outcome of D3A7 induction therapy. The FLT3-ITD mutation plays an important role in the 12-month survival of AML patients after D3A7 therapy. However, the outcome of D3A7 therapy and FLT3-ITD mutation were not associated with leukemia stem cells., (Copyright © 2024 Arwanih et al. This article is available under a Creative Commons License.)

Background: FLT3 gene mutations are genetic abnormality that caused leukemogenesis. Furthermore, presence of FLT3 mutations is associated with poor prognosis in AML. This study aimed to identify FLT3 gene mutations so that it can be used as a genetic reference for the AML patients in Indonesian population., Methods: This cross-sectional study recruited 63 AML de novo patients between August 2021 and July 2023 at Cipto Mangukusumo General Hospital and Dharmais Cancer Hospital. We collected peripheral blood from the patients for DNA isolation. FLT3 gene mutation was detected using PCR method, then followed by the Sanger sequencing. Novel mutation in exon-14 continued to in silico study using SWISS MODEL server for modelling protein and PyMOL2 software for visualizing the protein model., Results: Frequency FLT3-ITD mutation was 22% and 6 (10%) patients had a novel mutation on juxtamembrane domain. The number of FLT3-ITD insertions was 24 bp to 111 bp, with a median of 72 bp. Novel mutation indicated a change in the protein sequence at amino acid number 572 from Tyrosine to Valine and formed a stop codon (UGA) at amino acid position ins572G573. In-silico study from novel mutation showed the receptor FLT3 protein was a loss of most of the juxtamembrane domain and the entire kinase domain., Conclusion: A novel FLT3 gene mutation was found in this study in the juxtamembrane domain. Based on the sequencing analysis and in silico studies, this mutation is likely to affect the activity of the FLT3 receptor. Therefore, further studies on this novel mutation are needed., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Breast cancer stem cells (BCSCs) play a role in the high rates of resistance, recurrence, and metastasis. The precise biomarkers of BCSCs can assist effectively in identifying cancer, assessing prognosis, diagnosing, and monitoring therapy. The aim of this study was to give a complete analysis for predicting specific biomarkers of BCSCs., Methods: We aggregated profile datasets in this work to shed light on the underlying critical genes and pathways of BCSCs. We obtained two expression profiling by array datasets (GSE7513 and GSE7515) from the Gene Expression Omnibus (GEO) database to identify biomarkers in BCSCs. Enrichr was used to do functional analysis, including gene ontology (GO) and reactome pathway. Furthermore, the protein-protein interaction (PPI) of these differential expression genes (DEGs) was visualized using Cytoscape with the search tool for the retrieval of interacting genes (STRING). The hub genes in the PPI network were chosen for further investigation., Results: We identified 65 up-regulated and 190 down- regulated DEGs and the GO enrichment analysis revealed that these DEGs were enriched in biological process related to tumorigenesis and stemness, including alter the extracellular matrix's physicochemical properties, cytoskeletal reorganisation, adhesion, motility, migration, growth, and survival. The Reactome analysis indicated that these DEGs were also involved in modulating function of ECM, regulation cancer metabolism and angiogenesis, tumor growth, proliferation, and metastasis., Conclusion: Our bioinformatic study revealed that FYN, INADL, OCLN, F11R, and TOP2A were potential biomarker panel of BCSCs from secretome.

The aim of this study was to analyze the effect of heme synthesis inhibition on cytoglobin expression and its correlation with keloid fibroblast viability and proliferation. The study was conducted on primary culture of keloid fibroblasts. Heme synthesis in keloid fibroblasts was inhibited using succinyl acetone. We measured amino levulinic acid dehydratase (ALAD) enzyme activity using a colorimetric method; cytoglobin mRNA expression using qRT-PCR, cytoglobin protein expression using ELISA and immunocytochemistry, fibroblast viability using the MTT test; and fibroblast proliferation using BrdU test. The results showed that the ALAD enzyme activity level was lower in the keloid fibroblasts treated with succinyl-acetone (SA, 1, 2.5, and 5 mM) than in the control. The cytoglobin mRNA and protein expressions level were significantly lower in the keloid fibroblasts cultured with 2.5 mM and 5 mM SA than in the control and 1 mM SA. The viability and proliferation of the keloid fibroblasts decreased when the SA concentration was increased. In conclusion, the use of succinyl acetone at a concentration of 1; 2.5; and 5 mM caused decrease ALAD enzyme activity which indicated the inhibition of the heme synthesis. Inhibition of heme synthesis can affect cytoglobin expression, which correlates with the viability and proliferation of keloid fibroblasts., Competing Interests: No potential conflicts of interest were disclosed., (Copyright © 2023 JCBN.)

On Earth, there are significant variations in terms of exposure to naturally occurring radiation among different areas. Radon, a naturally-occurring radioactive gas that is the primary cause of lung cancer in nonsmokers and the second most prevalent cause among smokers, poses a considerable risk. Indoor radon, in particular, constitutes the most substantial source of natural radiation to which individuals are exposed. This study assessed the immune status of a population chronically exposed to high indoor radon concentration in Indonesia. Fifty-seven subjects from the Tande-Tande sub-village (high indoor radon concentration area) were compared to fifty-three participants living in the Topoyo village (low concentration area). We contrasted the immunological conditions of these two populations by measuring levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4), and IL-10 in serum. Moreover, we also measured levels of the nuclear factor kappa B (NF-κB), superoxide dismutase (SOD), glutathione peroxidase (GPX), and protein kinase B in its phosphorylated (pAkt) and non-phosphorylated form (Akt) in peripheral blood mononuclear cells (PBMCs) of a subset of participants (31 from each population). TNF-α, IFN-γ, and IL-4 levels in Tande-Tande sub-village inhabitants were significantly lower than those in the control group living in the Topoyo village (p = 0.001, p = 0.017, and p = 0.002). The concentration of IL-10 also tended to be lower in people living in the high indoor radon concentration area, but it did not differ significantly between Tande-Tande sub-village inhabitants and Topoyo inhabitants (p = 0.106). Protein levels of NF-κB, pAkt, and Akt in Tande-Tande sub-village inhabitants also did not differ significantly between Tande-Tande sub-village inhabitants and Topoyo inhabitants (p = 0.234, p = 0.210, and p = 0.657). Similarly, activities of SOD and GPX did not differ significantly between the two populations (p = 0.569 and p = 0.949). Overall, despite their chronic exposure to high indoor radon concentrations, our study revealed no increase in the levels of TNF-α, IFN-γ, IL-10, IL-4, SOD, and GPX in the inhabitants of Tande-Tande sub-village compared with people living in the Topoyo village. Furthermore, our study demonstrated no activation in the Akt pathway, as indicated by the pAkt/Akt ratio observed in PBMC lysates of individuals residing in the Tande-Tande sub-village., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The frequencies of unstable and stable chromosome aberrations and micronuclei were examined in peripheral blood samples from 10 individuals living in elevated radon concentration areas (Takandeang Village, Mamuju, Indonesia). Blood samples from 10 people living in Topoyo Village were used as a control group. For unstable chromosome aberration analysis, a dicentric chromosome assay was conducted using conventional Giemsa staining. Chromosomal painting of chromosomes 1 and 4 using the fluorescence in situ hybridisation technique was also applied to four subjects to assess the stable chromosome aberration. Our study showed no significant increases across all groups in dicentric and other unstable chromosome aberrations, such as rings and acentric fragments. Translocations were found in one person from Takandeang Village and two Topoyo Village inhabitants. The translocations found in the subjects from Takandeang Village were due more to aging factors than to radon exposure. The number of micronuclei per 1000 binucleus cells in Takandeang Village inhabitants was not significantly different than that in the control group (p = 0.943). A more comprehensive analysis should be conducted in a subsequent study by increasing the number of study donors and the number of metaphases to be analysed in both dicentric chromosome assay and fluorescence in situ hybridisation assays. Such research could provide valid information on the cytogenetic effects of elevated indoor radon exposure., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: The aim of this study was to investigate the effect of mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) on the human MCF7 breast cancer cell proliferation that have been considered to contain limited CSC population and its association with the expression of OCT4 and ALDH1 stemness markers., Methods: EVs were successfully isolated from the conditioned medium of umbilical cord MSCs using size exclusion chromatography. The isolated EV fraction was verified under a transmission electron microscope (TEM). Five and ten percent (v/v) concentration of MSC-EVs were then co-cultured with MCF7 cells. To investigate MSC-EV uptake by MCF7 cells, we performed confocal microscopy analysis. Subsequently, the proliferation of co-cultured MCF7 cells was determined using trypan blue exclusion assay, while their mRNA and protein expression of OCT4 as well as ALDH activity as the marker of stemness properties were analyzed using quantitative reverse transcription polymerase chain reaction, Western Blot, and Aldefluor™ assays, respectively., Result: MSC-EVs were detected as round-shaped, ~100 nm sized particles under TEM. We also demonstrate that MSC-EVs can be internalized by MCF7 cells. Notably, MSC-EVs of 5% concentration increased OCT4 mRNA expression and ALDH1 activity in MCF7 cells. At 10% concentration, MSC-EVs reduced the OCT4 expression and ALDH1 activity., Conclusion: MSC-derived EVs modulate the stemness of MCF7 cells, either OCT4 expression or ALDH1 activity, in a concentration dependent manner along with the increase of cell proliferation.

Purpose: To deepen our knowledge on the effects of high levels of indoor radon exposure, we assessed the frequencies of unstable and stable chromosome aberrations and micronucleus (MN), as well as the concentration of an endogenous antioxidant (catalase, CAT), in blood samples of individuals chronically exposed to high indoor radon concentrations in Indonesia (Tande-Tande sub-village, Mamuju, West Sulawesi). Moreover, we also investigated the occurrence of a radio-adaptive response (RAR) in Tande-Tande sub-village inhabitants using the G 2 MN assay., Materials and Methods: The frequencies of dicentric (DC), acentric (AF), ring (R), and translocation (Tr) chromosomes in Tande-Tande inhabitants were compared to those in people living in a reference area with low levels of indoor radon levels (Topoyo village, Indonesia). The number of MN per 1000 binucleated cells (BNC) and CAT concentration per total protein was quantified and compared between groups. Lastly, we irradiated (2 Gy) phytohemagglutinin-stimulated samples in vitro and measured the frequency of MN to verify the occurrence of a RAR in Tande-Tande sub-village inhabitants., Results and Conclusion: The frequencies of DC, AF, and Tr did not differ between Tande-Tande inhabitants and control subjects ( p  = 0.350, 0.521, 0.597). The frequency of MN in Tande-Tande inhabitants was significantly lower than that in the control group ( p  = 0.006). Similarly, CAT concentration in Tande-Tande inhabitants was also significantly lower than that in the control population ( p  < 0.001). Significant negative correlations were identified for MN number and CAT concentration versus indoor radon concentration, annual effective dose, or cumulative dose both within groups and when all data were analyzed together. Our findings indicate that, despite the high indoor radon levels, Tande-Tande inhabitants are not under oxidative stress, since this group had lower CAT concentration and MN frequency than those in the control group. The negative correlation between MN frequency and indoor radon concentration, annual effective dose, and cumulative dose suggests the occurrence of an RAR phenomenon in Tande-Tande sub-village inhabitants. This interpretation is also supported by the results of the G 2 MN assay, which revealed lower MN frequencies after in vitro irradiation of samples from Tande-Tande sub-village inhabitants than those in samples from the control group ( p  = 0.0069, for cumulative MN frequency; p =  0.0146, for radiation-induced MN only).

Acute myeloid leukemia (AML) is a hematological malignancy commonly found in adult patients. Low overall survival and resistance to therapy are the main issues in AML. The first line of treatment for AML chemotherapy is the induction phase, namely, the phase to induce remission by administering a combination of daunorubicin (DNR) for three days followed by administration of cytarabine (Ara-C) with continuous infusion for seven days, which is referred to as "3 + 7." Such induction therapy has been the standard therapy for AML for the last four decades. This review article is made to discuss daunorubicin and cytarabine from their chemical structure, pharmacodynamics, pharmacokinetics, and mechanisms of resistance in AML., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Arwanih et al.)

The radiation response of cervical cancer is thought to be enhanced by the levels of melatonin due to its roles in the circadian cycle and cancer growth. In the present study, the roles of circadian rhythms and melatonin levels as prognostic factors for predicting the radiation response in patients with cervical cancer were examined. In this nested case‑control study, patients with good and poor responses to radiotherapy were assessed in terms of the time‑of‑day radiation treatment was administered and further influencing factors. The radiation time was determined, as the subjects were either irradiated in the morning (06.00‑10.00 am) or afternoon (04.00‑06.00 pm). Data on tumour size and other biological parameters were collected and analysed by binary logistic regression. Among the 56 patients examined, most subjects had good radiation responses. Most patients were <50 years old with an initial body weight of >50 kg, no pain prior to radiation, low erythrocyte sedimentation rates, normal intravenous urography results, moderate or good differentiation on pathology and histo‑pathologically non‑keratinised cells. According to the multivariate analysis, the irradiation time as a surrogate of the circadian cycle (morning vs. afternoon), the initial haemoglobin (Hb) level and the clinical tumour size were significant predictors of the radiation response. The circadian cycle, tumour size and Hb levels may affect the radiation response in patients with cervical cancer. In addition, the morning group had better 5‑year overall survival, but it was not significant, possibly due to the small cohort size. Further research is required to identify more relevant prognostic factors using different radiotherapy techniques [National Clinical Trial (NCT) no. NCT05511740, registration date, 08/20/2022].

Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also toxic for normal cell, not specific to the target cells. Consequently, chemotherapy has various side effects. Monoclonal antibody (MAb) has been developed for specific therapy which only has killing effect in cancer cells, but the survival rate of most MAbs around 20%. Therefore, in clinical practice, MAbs administration should combine with chemotherapeutic agents. For effectiveness of therapy and to minimalize adverse effects, anticancer agent with selective cytotoxic effect on target cells is needed, the immunotoxin., Objective: This study introduces a novel approach to conjugate monoclonal antibody (Cetuximab) and toxin (Puromycin), in order to selectively inhibit proliferation of triple negative breast cancer (TNBC) and to enhance the efficacy of MAb in target cells killing., Methods: Cetuximab was conjugated with Puromycin using a linker, i.e SATP (Succinimidyl-acetylthiopropionate) and tested on triple negative breast cancer cell lines (MDA-MB-231) which expressed EGFR (epidermal growth factor receptor). Cetuximab is MAb which targets EGFR. MCF-7 was used as control cells since it has low or no EGFR expression. Cell counting were conducted as viability assay at 24 hours, 48 hours, and 72 hours after treatment., Results: The results showed significant reduction of live cells number in Conjugate 20 µg/mL cultured in MDA-MB-231 compared to MCF-7 after 24 hours, 48 hours, and 72 hours incubation. In all time period of incubation, significant reduction of MDA-MB-231 live cells number was also observed in Conjugate 20 µg/mL compared to Cetuximab 20 µg/mL., Conclusion: Synthesized conjugate showed its target-specific effect in TNBC and improved the efficacy of Cetuximab on TNBC. In the future, this conjugate can be a potential anticancer therapy in treating triple-negative breast cancer.

The objective of this study was to assess the effect of radon exposure on the modulation of endogenous antioxidants in a population chronically exposed to high levels of radon indoors. To do so, we measured the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in peripheral blood mononuclear cells (PBMCs) of people living in an area with high indoor radon concentration (Tande-Tande sub-village, Indonesia). The activities of SOD and GPX in Tande-Tande inhabitants were compared with those in subjects living in the Topoyo village (Indonesia), an area with low indoor radon levels. The activities of SOD and GPX in Tande-Tande sub-village inhabitants did not differ from those in people living in the Topoyo village (0.37 ± 0.021  versus 0.28 ± 0.018 U/mg protein and 8.46 ± 1.48 versus 8.34 ± 1.65 U/mg protein, p  > .05). For both populations, there was a significant positive correlation between SOD and GPX activities ( p  < .001). No significant effects of gender, age, smoking habit, and body mass index on SOD and GPX activities were found for both groups. Although no significant modulation of SOD and GPX activities in PBMCs was detected, further studies should expand the sample size and also assess antioxidant levels in the serum. This study provides a first picture of endogenous antioxidant systems in Tande-Tande sub-village inhabitants, but a more comprehensive analysis, including the measurement of catalase (CAT) activity, might provide additional insight into the effects of chronic exposure to high indoor radon concentrations.

Cell signaling is a vital part of biological life. It helps coordinating various cellular processes including cell survival, cell growth, cell death, and cell interaction with the microenvironment and other cells. In general, cell signaling involves the attachment of signaling molecules known as ligands to specific receptors on cell surface, which then activate downstream events that dictate the cell's response. One of the most studied ligands is transforming growth factor-beta (TGF-β). TGF-β signaling is mainly mediated by suppressor of mothers against decapentaplegic (Smad) proteins, but it also interacts with other pathways such as the Ras and mitogen-activated protein kinase (MAPK) signaling pathways. Furthermore, TGF-β can have a dual role depending on the cellular and microenvironmental context, in which it can act as either a growth promoter or a growth inhibitor. It has been known that TGF-β can self-induce its ligand production, thereby prolonging and amplifying its effect on cells and their microenvironment. The aim of this review is to discuss the regulation and signaling of TGF-β autoinduction, which still remain to be elucidated. Several factors have been found to facilitate TGF-β autoinduction, which include the activator protein-1 (AP1) complex, Smad3-dependent signaling, and non-Smad signaling pathways. On the other hand, the LIM (Lin11, Isl-1 and Mec-3) domain only 7 (LMO7) protein can suppress TGF-β autoinduction by interfering with the activities of AP-1 and Smad3. Since TGF-β autoinduction is implicated in various pathological conditions, better understanding of its regulation and signaling can provide new directions for therapy., Competing Interests: The authors declare no competing interests.

Objective: The aim of this study was to investigate the effect of cancer-associated fibroblasts (CAF) secretomes on the epithelial-mesenchymal transition (EMT) of colorectal carcinoma (CRC) cells and its association with hepatocyte growth factor (HGF) signalling focussing on the HGF receptor, c-Mesenchymal epithelial transition (c-Met), and the EMT markers, vimentin and e-cadherin, in CRC cells., Methods: Conditioned mediums (CM) containing secretomes from colorectal CAFs and their counterpart normal fibroblasts (NFs) of three CRC patients were collected and supplemented to the HT-29 CRC cells. The mRNA levels of a-smooth muscle actin (a-SMA) and HGF in both fibroblasts, as well as c-Met, vimentin, and e-cadherin in HT-29 cells after supplemented with CAF- and NF-CM were determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). HGF protein level in the CM of CAFs and NFs was measured using enzyme-linked immunosorbent assay (ELISA). Vimentin and e-cadherin protein expressions were observed in HT-29 cells using immunofluorescent (IF) staining., Results: Compared to the non-cancerous colon, fibroblasts from cancerous area of CRC substantially expressed higher mRNA levels of a-SMA, a CAF marker. The HGF mRNA expressions in CAFs and NFs were in line with the HGF protein level in the secretomes of both cells. CAF-CM increased c-Met and vimentin mRNA levels in HT-29 cells. Surprisingly, e-cadherin mRNA level in HT-29 cells was increased following CAF-CM supplementation. We also demonstrated the co-localization of e-cadherin and vimentin in the HT-29 cell cytoplasm., Conclusions: CAF secretomes of CRC promote a hybrid type of EMT associated with HGF signalling.

Objective: To investigate the auto-induction of transforming growth factor-b1 (TGF-β1) in breast cancer stem cells (BCSCs) and its effect on cell viability and stemness., Methods: Human BCSCs (aldehyde dehydrogenase positive; ALDH+) were grown in serum-free Dulbecco's Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12) and treated for periods of 1, 2 and 4 hours with 0.1 ng/ml recombinant human TGF-β1 protein (rhTGF-β1). The medium was then replaced with serum-free DMEM/F12 without rhTGF-β1 for 24 hours. Cell viability was determined using a trypan blue exclusion assay. Type 1 TGF-β receptor (TβR1), TGF-β1, octamer-binding transcription factor 4 (OCT4) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) messenger RNA (mRNA) expression levels were analysed using quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR). The TGF-β protein level in the culture medium was determined using an enzyme-linked immunosorbent assay (ELISA)., Results: The expression levels of rhTGF-β1, TGF-β1 and TβR1 mRNA significantly increased in BCSCs compared to control after treatment for 1 and 2 hours but decreased after 4 hours. This is in line with alteration of stemness gene, OCT4 and ALDH1A1 mRNA expressions. However, the secretion of newly synthesised TGF-β1 significantly increased after 2 hours. In contrast, viable BCSCs decreased after 1 hour and then gradually increased 2.7 times compared to control after 4 hours., Conclusions: TGF-β1 treatment in low concentration and for short period of time triggers its auto-induction in BCSCs, leading to increased cell viability and stemness gene expression via autocrine signalling.

Breast cancer stem cells (BCSCs) express high levels of the anti-apoptotic protein, survivin. This study aimed to discover a natural active compound with anti-cancer properties that targeted survivin in human breast cancer stem cells. From the seven examined compounds, andrographolide was selected as a lead compound through in silico molecular docking with survivin, caspase-9, and caspase-3. We found that the affinity between andrographolide and survivin is higher than that with caspase-9 and caspase-3. Human CD24-/CD44+ BCSCs were treated with andrographolide in vitro for 24 hours. The cytotoxic effect of andrographolide on BCSCs was compared to that on human mesenchymal stem cells (MSCs). The expression of survivin, caspase-9, and caspase-3 mRNA was analyzed using qRT-PCR, while Thr34-phosphorylated survivin and total survivin levels were determined using ELISA and Immunoblotting assay. Annexin-V/PI flow cytometry assays were performed to evaluate the apoptotic activity of andrographolide. Our results demonstrate that the CC50 of andrographolide in BCSCs was 0.32mM, whereas there was no cytotoxic effect in MSCs. Moreover, andrographolide decreased survivin and Thr34-phosphorylated survivin, thus inhibiting survivin activation and increasing survivin mRNA in BCSCs. The apoptotic activity of andrographolide was revealed by the increase of caspase-3 mRNA and protein, as well as the increase in both the early and late phases of apoptosis. In conclusion, andrographolide can be considered an anti-cancer compound that targets BCSCs due to its molecular interactions with survivin, caspase-9, and caspase-3, which induce apoptosis. We suggest that the binding of andrographolide to survivin is a critical aspect of the effect of andrographolide., Competing Interests: The authors have declared that no competing interests exist.

Purpose: This study was intended to find out the impact of alpha mangostin administration on the epithelial-mesenchymal transition (EMT) markers and TGF-β/Smad pathways in hepatocellular carcinoma Hep-G2 cells surviving sorafenib. Methods: Hepatocellular carcinoma HepG2 cells were treated with sorafenib 10 μM. Cells surviving sorafenib treatment (HepG2 surv ) were then treated vehicle, sorafenib, alpha mangostin, or combination of sorafenib and alpha mangostin. Afterward, cells were observed for their morphology with an inverted microscope and counted for cell viability. The concentrations of transforming growth factor (TGF)-β1 in a culture medium were examined using ELISA. The mRNA expressions of TGF-β1, TGF-β1-receptor, Smad3, Smad7, E-cadherin, and vimentin were evaluated using quantitative reverse transcriptase-polymerase chain reaction. The protein level of E-cadherin was also determined using western blot analysis. Results: Treatment of alpha mangostin and sorafenib caused a significant decrease in the viability of sorafenib-surviving HepG2 cells versus control (both groups with P <0.05). Our study found that alpha mangostin treatment increased the expressions of vimentin ( P <0.001 versus control). In contrast, alpha mangostin treatment tends to decrease the expressions of Smad7 and E-cadherin (both with P >0.05). In line with our findings, the expressions of TGF-β1 and Smad3 are significantly upregulated after alpha mangostin administration (both with P <0.05) versus control. Conclusion: Alpha mangostin reduced cell viability of sorafenib-surviving HepG2 cells; however, it also enhanced epithelial-mesenchymal transition markers by activating TGF-β/Smad pathways., (© 2020 The Authors.)

Background: Various chemical agents have been used as an adjuvant treatment for giant cell tumor (GCT). However, the comparative effect of these chemicals remains unclear., Methods: Multinucleated and spindle cells from cultured GCT patients, characterized by Nanog and Oct4 expression with RT-PCR, were directly administered, in vitro, with concentrations of 1%, 3%, and 5% of H 2 O 2 and 75%, 85%, and 95% of ethanol for 10 minutes and concentrations of 0.003%, 0.005%, 0.01%, 0.03%, 0.1%, and 0.3% of H 2 O 2 for 5 minutes and were incubated for 24 hours. Cell morphology, cell viability, and flow cytometry after various concentrations of H 2 O 2 and ethanol exposure were assessed., Results: H 2 O 2 in all concentrations caused loss of cell viability. The number of viable cells after H 2 O 2 exposure was related to the concentration-dependent effect. The initial viable spindle-shaped cell, multinucleated giant cell, and round-epithelioid cell had morphological changes into fragmented nonviable cells after exposure to H 2 O 2 . Flow cytometry using Annexin V showed cell death due to necrosis, with the highest concentration amounting to 0.3%., Conclusion: Administering local chemical adjuvants of H 2 O 2 in vitro caused loss of viable GCT cells. The number of viable cells after H 2 O 2 exposure was related to the concentration-dependent effect, whereas reducing concentration of H 2 O 2 may cause loss of viability and morphology of cultured GCT cells with the apoptotic mechanism., Competing Interests: Achmad Fauzi Kamal, Akbar Rizki Beni Asdi, Rahyussalim, Rio Wikanjaya, Resda Akhra Syahrani, Tri Kurniawati, and Septelia Inawati Wanandi declare that they have no conflict of interest., (Copyright © 2020 Achmad Fauzi Kamal et al.)

Background: Despite recent progress in molecular-targeted therapies, breast cancer remains the primary leading cause of cancer related death among women worldwide. Breast cancer stem cells (BCSCs) are believed to be responsible for therapy resistance and cancer recurrence. We recently demonstrated that human BCSCs (CD24-/CD44+) could survive better than their counterpart non-BCSCs (CD24-/CD44-) when treated with rotenone, possibly due to lower levels of reactive oxygen species (ROS) production, high expression of antioxidant manganese superoxide dismutase (MnSOD), and anti-apoptotic survivin. The aim of this study was to verify the role of survivin on human BCSCs survival under oxidative stress modulation by suppressing its expression using YM155, a survivin inhibitor., Methods: Human BCSCs (ALDH+ cells) were treated with YM155 for 24 h prior to treatment with rotenone for a further 6 h. We determined intracellular superoxide levels were determined using dihydroethidium assay, survivin and MnSOD expression using qRT-PCR, survivin protein level using ELISA, as well as cell viability using trypan blue exclusion and acridine orange/ethidium bromide apoptosis assay., Results: Suppression of survivin expression using YM155 could reduce the survival of rotenone-treated BCSCs, which may be associated with oxidative stress modulation, as shown by increased ROS levels and decreased MnSOD expression. We confirm that survivin is responsible for maintaining BCSCs survival under oxidative stress modulation. Furthermore, YM155 could modulate oxidative stress in BCSCs by reducing MnSOD expression and increasing ROS levels., Conclusion: YM155 treatment could be used to overcome BCSCs resistance to oxidative stress-based anticancer therapies.

Background: Platelet-rich plasma (PRP) contains pro-angiogenic growth factors including vascular endothelial growth factor (VEGF). Angiogenesis is a necessary component of wound healing in instances of diabetic foot ulcers (DFU). PRP composition varies depending on methods and donor health status. Our group has developed an improved PRP protocol for diabetes treatment. The aims of this study were to examine the levels of the pro-angiogenic factor VEGF in these patient populations with and without diabetes., Methods: PRP was prepared using 24 mL of whole blood from 13 diabetic and 10 non-diabetic patients registered at Klinik Hayandra. Whole blood in sodium citrate tubes were centrifuged at 1,000 rpm for 5 minutes followed by plasma separation. Plasma samples were centrifuged at 3,000 rpm for 5 minutes. Upper platelet-poor plasma layers were discarded, leaving 5 mL of concentrated platelet containing plasma (PRP). Concentrated plasma samples were mixed, aliquoted, stored at -86 °C, and pooled for platelet count, VEGF, and total protein analyses. Platelet counting was also performed using fresh whole blood and PRP to measure changes following PRP preparation., Results: Diabetic donors had higher whole blood platelet counts than non-diabetic donors, but this difference was not statistically significant. An average increase of more than 250% in platelet number after PRP preparation using our method was noted in both groups. Freezing-thawing samples at -86 °C lysed more than 90% of PRP platelets regardless of diabetes status. Diabetic PRP had lower mean total protein and higher VEGF concentrations. Lysed platelets from diabetic donors released more VEGF than those from non-diabetic donors., Conclusions: PRP from diabetic donors had higher VEGF content making autologous PRP application a promising treatment for DFU. However, this should be investigated another appropriate clinical trial., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare., (2019 Stem Cell Investigation. All rights reserved.)

Objective: This study aimed to identify the association between duration of HU administration prior to IM treatment and MMR achievement in chronic-phase CML while evaluating the role of MDA, HIF-1α and P-gp., Methods: The study was conducted at Dr. Cipto Mangunkusumo National General Hospital and Dharmais Cancer Hospital, Jakarta using retrospective cohort design to analyse the association between the duration of HU before IM and its MMR achievement and cross-sectional design to analyse the association between MDA, HIF-1α and P-gp expressions with MMR achievement. Main subjects were chronic-phase CML patients treated by HU prior to IM for ≥ 12 months and HU only. The subjects were divided into four main groups: (1) chronic-phase CML patients treated with HU ≤ 6 months + IM ≥ 12 months and (2) HU > 6 months + IM ≥ 12 months (3) HU only (≤ 6 months), (4) HU only ( >6 months). Subjects were obtained from January 2015 to May 2016. Data were gathered through history taking, physical examination, medical record evaluation, and blood sample analysis. Bivariate analysis was conducted using chi square, independent T-test, and Mann-Whitney according to the variables., Results: Administration of HU for more than 6 months prior to IM was associated with unsuccessful MMR achievement (RR 1.60; 95%CI 1.29-2.00). MDA level, HIF-1α, P-glycoprotein expression were not associated with MMR achievement but the mean MDA level (0.63±0.31 vs 0.75±0.41 p=0.461) and median P-glycoprotein expressions {16,92 (0,04 - 43,86) vs. 5,15 (0,02-39,64); p=0.311} were found to be higher in patients receiving HU for > 6 months group than in HU ≤ 6 months group consecutively., Conclusion: Administration of HU for more than 6 months prior to IM was associated with unsuccessful MMR achievement in chronic-phase CML. The study suggested that P-glycoprotein overexpression as the predictor for unsuccessful MMR achievement.

Background: Glioblastoma multiforme (GBM) is the most malignant primary brain tumour and there is no definite cure. It has been suggested that there are significant interactions among mesenchymal stem cells (MSCs), their released factors and tumour cells that ultimately determine GBM's growth pattern. This study aims to analyse the expression of molecules involved in GBM cell apoptotic pathways following treatment with the MSC secretome., Methods: A conditioned medium of umbilical cord-derived MSCs (UCMSC-CM) was generated by culturing the cells on serum-free αMEM for 24 h. Following this, human GBM T98G cells were treated with UCMSC-CM for 24 h. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was then performed to measure the mRNA expression of survivin, caspase-9, TNF-related apoptosis-inducing ligand (TRAIL), DR4 and DcR1., Results: mRNA expression of caspase-9 in CM-treated T98G cells increased 1.6-fold ( P = 0.017), whereas mRNA expression of survivin increased 3.5-fold ( P = 0.002). On the other hand, TRAIL protein expression was upregulated (1.2-fold), whereas mRNA expression was downregulated (0.4-fold), in CM-treated cells. Moreover, there was an increase in the mRNA expression of both DR4 (3.5-fold) and DcR1 (1,368.5-fold) in CM-treated cells., Conclusion: The UCMSC-CM was able to regulate the expression of molecules involved in GBM cell apoptotic pathways. However, the expression of anti-apoptotic molecules was more upregulated than that of pro-apoptotic molecules., Competing Interests: Conflict of Interest None., (© Penerbit Universiti Sains Malaysia, 2019.)

Background: It has been widely reported that breast cancer aggressiveness may be driven by breast cancer stem cells (BCSCs). BCSCs display stemness properties that include self-renewal, tumourigenicity and pluripotency. The regulation of gene expression may have important roles in BCSC stemness and aggressiveness. Thus, the aim of this study was to examine the stemness and aggressiveness gene expression profile of BCSCs compared to MCF-7 and MDA-MB-231 breast cancer cells., Methods: Human ALDH1+ BCSCs were grown in serum-free Dulbecco's Modified Eagle Medium (DMEM)/F12, while MCF-7 and MDA-MB-231 were cultured in DMEM supplemented with 10% foetal bovine serum under standard conditions. Total RNA was extracted using the Tripure Isolation Reagent. The relative mRNA expressions of OCT4, ALDH1A1 and CD44 associated with stemness as well as TGF-β1, TβR1, ERα1 and MnSOD associated with aggressiveness in BCSCs and MCF-7 cells were determined using the quantitative real-time PCR (qRT-PCR)., Results: The mRNA expressions of OCT4 (5.19-fold ± 0.338; P = 0.001), ALDH1A1 (3.67-fold ± 0.523; P = 0.006), CD44 (2.65-fold ± 0.307; P = 0.006), TGF -β 1 (22.89-fold ± 6.840; P = 0.015), TβR1 (3.74-fold ± 1.446; P = 0.045) and MnSOD (4.6-fold ± 1.096; P = 0.014) were higher in BCSCs than in MCF-7 but were almost similar to MDA-MB-231 cells. In contrast, the ERα1 expression of BCSCs (0.97-fold ± 0.080; P = 0.392) was similar to MCF-7 cells, indicating that BSCSs are oestrogen-dependent breast cancer cells., Conclusion: The oestrogen-dependent BCSCs express stemness and aggressiveness genes at a higher level compared to oestrogen-dependent MCF-7 but are almost similar to oestrogen-independent MDA-MB-231 cells., Competing Interests: Conflict of Interest None., (© Penerbit Universiti Sains Malaysia, 2019.)

Cancer stem cells (CSCs) are a subpopulation of cancer cells responsible for tumor maintenance and relapse due to their ability to resist various anticancer effects. Owing to the resistance of CSCs to the effects of targeted therapy, an alternative strategy that targets post-translational glycosylation may be an improved approach to treat cancer as it disrupts multiple coordinated signaling that maintains the stemness of CSCs. Glucosamine acts as an anticancer agent possibly by inhibiting N-linked glycosylation. The aim of the present study was to investigate the effect of glucosamine on the stemness of breast CSCs, which is regulated by signal transducer and activator of transcription 3 (STAT3) signaling. Human aldehyde dehydrogenase-positive (ALDH + ) breast CSCs and MCF7 cells were treated with various concentrations (0.25, 1 or 4 mM) of glucosamine for 24 h. Subsequently, cell viability was determined by performing a trypan blue exclusion assay, pluripotency gene [ALDH 1 family member A1 ( ALDH1A1 ), octamer-binding transcription factor 4 ( OCT-4 ), and Krüppel-like factor 4 ( KLF4 )] expression was determined using the reverse transcription-quantitative polymerase chain reaction, and STAT3 and phosphorylated STAT3 (pSTAT3) levels were determined by performing western blot analysis. Furthermore, the number of mammosphere-forming units (MFUs) in ALDH + breast CSCs and MCF7 cells was determined. It was determined that glucosamine treatment decreased the viability of ALDH + breast CSCs. Glucosamine treatment also decreased the stemness of ALDH + breast CSCs and MCF7 cells, as indicated by decreased ALDH1A1, OCT-4 and KLF4 expression level, and a decreased number of MFUs. This effect of glucosamine may be associated with a decreased pSTAT3/STAT3 ratio, indicating that glucosamine inhibited STAT3 activation; therefore, the results of the present study indicated that glucosamine treatment may be an improved approach to target the stemness of CSCs.

Background: Neuroglobin (Ngb) is highly expressed in the suprachiasmatic nucleus, and can regulate Per1 gene expression. It is still not known whether Ngb also influences Cryptochrome (Cry). Cry is implicated in hypertension and primary aldosteronism (PA) in mice. There is a strong correlation between Obstructive Sleep Apnea (OSA) and PA. We propose to prove that Ngb and Cry play a role in OSA with PA., Methods: Subjects were recruited consecutively from residents of Jakarta, Indonesia; subjects aged 30-65 years with moderate to severe OSA and hypertension were included in the study. OSA was diagnosed using an unattended type 2 portable monitor (Alice Pdx), hypertension was diagnosed when morning blood pressure exceeded 140/90 mmHg or when taking anti-hypertensive drugs. Serum concentration of aldosterone, renin, Cry1, Cry2 and Ngb protein were determined using ELISA method. Primary aldosteronism (PA) was defined as ARR ≥20., Results: Forty subjects were recruited, 26 male and 14 female, median age 52.5 years, BMI 27.46 kg/m2, and AHI 34.8 times/hour. We found 16 subjects with PA and 24 non PA. Cry1 and Cry2 did not correlate with ARR in PA and non PA groups. Ngb correlated positively with Cry1 (Spearman's rho = 0.455, p = 0.038) but not Cry2 in PA patients. Cry1 concentration decreased in severe hypoxia., Conclusions: Ngb correlates with Cry1 in OSA with PA. There is no correlation between Cry1 or Cry2 with PA., Competing Interests: The authors have declared that no competing interests exist.

A cross-sectional study on hepatitis B patients in Indonesia showed association of pre-S2 start codon mutation (M120 V) with cirrhosis and hepatocellular carcinoma (HCC), which was dissimilar from studies from other populations where pre-S2 deletion mutation was more prevalent. Different mutation patterns were attributed to different hepatitis B virus (HBV) subgenotypes in each population study. HBV surface proteins are reported to induce the activation of NF-κB, a transcriptional factor known to play an important role in the development of liver disease. This study aimed to see the effects of HBs variants in HBV subgenotype B3 on the expression and activation of NF-κB as one of the mechanisms in inducing advanced liver disease. HBV subgenotypes B3, each carrying wild-type (wt) HBs, M120 V, and pre-S2 deletion mutation were isolated from three HCC patients. HBs genes were amplified and cloned into pcDNA3.1 and were transfected using Lipofectamine into a Huh7 cell line. NF-κB activation was measured through IκB-α expression, which is regulated by NF-κB. RNA expressions for HBs, IκB-α, and NF-κB subunit (p50) were evaluated using real-time PCR. M120 V mutant had a significantly higher mRNA level compared with wt and pre-S2 deletion mutant; however, there were no significant differences in HBs protein expressions. The transcription level of p50 was higher in M120 V mutation compared with HBs wild-type and pre-S2 deletion mutant. NF-κB activation was higher in HBs wild-type compared with the two mutant variants. Pre-S2 mutations had no effect on the increment of NF-κB activation. However, M120 V mutation may utilize a different pathway in liver disease progression that involves high expression of NF-κB subunit, p50.

Background: Curcumin was shown to reduce epithelial-mesenchymal transition (EMT) markers in previous short term studies. This study was aimed to investigate the potential of curcumin in the prevention of EMT activation in MCF-7 cells induced by endoxifen. Methods: MCF-7 breast cancer cells were treated with Endoxifen 1000 nM+betaestradiol 1 nM with or without curcumin (8.5μM or 17 μM). Cells treated with dimethyl sulfoxide (DMSO) 0.001% were used as negative control. After 8 weeks of continuous treatment, the cells were counted, analyzed for mRNA E-cadherin, vimentin, TGF-β expression, total reactive oxygen species (ROS) and observed for morphological changes using confocal microscope and transmission electron microscope. Result: MCF-7 cell viability was increased in endoxifen + β-estradiol group. Cell viability was significantly decreased in curcumin 17 μM, but not in curcumin 8.5 μM group. Analysis of EMT markers at week 8 indicates that there were increase in vimentin and TGF-β mRNA expressions, while E-cadherin mRNA expressions and TGF-β1 protein concentrations were shown to decrease. The results showed that administration of curcumin in all the dose administered were incapable improving the expressions of vimentin, TGF-β1 and E-cadherin. There was a decrease in ROS concentration in curcumin treated cells (8.5 μM) while in curcumin 17 μM, ROS concentration was increased. Morphological observation using confocal microscope and TEM showed the presence of mesenchymal cells and adherens junction. Conclusion: endoxifen treatments for eight weeks resulted in upregulation of EMT markers and changes in morphology of MCF-7 breast cancer cells. The addition of curcumin did not prevent the activation of EMT., (Creative Commons Attribution License)

The growth of tumour cells is closely related to cancer-associated fibroblasts (CAFs) present within their microenvironment. CAFs, the most abundant cells in tumour stroma, secrete growth factors that play pivotal roles in tumour cell proliferation, metabolism, angiogenesis and metastasis. Tumour cells adapt to rapid environmental changes from normoxia to hypoxia through metabolic interplay with CAFs. In this mini review, we discuss the role of lactate dehydrogenases (LDHs) and monocarboxylate transporters (MCTs) on the metabolic interplay between tumour cells and CAFs under hypoxia compared to normoxia. The LDHs catalyse the interchange of lactate and pyruvate, whereas MCTs facilitate the influx and efflux of monocarboxylates, especially lactate and pyruvate. To sum up, tumour cells switch their metabolic state between glycolysis and oxidative phosphorylation through metabolic interplay with CAFs, which exhibit the Warburg effect under hypoxia and reverse Warburg effect under normoxia.

Cancer stem cells reside in a distinct region within the tumor microenvironment that it is believed to play a fundamental role in regulating stemness, proliferation, survival, and metabolism of cancer cells. This study aimed to analyze the effect of extracellular alkalinization on metabolism and survival of human CD24-/CD44+ breast cancer stem cells (BCSCs). BCSCs were cultured in alkalinized DMEM-F12 and incubated at 37°C, 5% CO2, and 20% O2 for 30 min, 6, 24, and 48 h. After each incubation period, we analyzed the modulation of various mRNA expressions related to pH and cellular metabolic regulation using the qRT-PCR. Metabolic state was measured using colorimetric and fluorometric assays. To examine cell proliferation and apoptosis, we used trypan blue and annexin V/propidium iodide assay, respectively. This study demonstrated that alkalinization could stimulate extracellular carbonic anhydrase (CAe) activity, as well as CA9 and HIF1α expression. Under alkaline pH and HIF1α regulation, glucose consumption, extracellular lactate production, and LDH activity of BCSCs were upregulated while O2 consumption was downregulated. These metabolic shifts seemed to promote apoptosis and suppress the proliferation of BCSCs. To conclude, modulation of the extracellular environment through alkalinization could change the metabolic states of BCSCs, which in turn affect the cell survival.

Oxidative stress is considered an important factor in the development of endometriosis, including its malignant transformation. Previous studies have found that AT-rich interactive domain 1A ( ARID1A ), a tumor suppressor gene, is frequently mutated and inactivated in endometriosis-associated ovarian cancer (EAOC), and such a change in this gene is considered an early event in malignant transformation. We observed oxidative stress status by measuring the activity of the antioxidant enzyme manganese superoxide dismutase (MnSOD), malondialdehyde (MDA), and ARID1A gene expression in tissue samples from patients with endometriosis, EAOC, or non-endometriosis-associated ovarian cancer (non-EAOC). We also induced oxidative stress in the cultured cells from patients with primary endometriosis by adding H 2 O 2 and tested for any alteration of ARID1A gene expression based on different H 2 O 2 concentrations. The results showed that MnSOD activity in endometriosis and EAOC was lower than in non-EAOC, but MDA levels were higher. This study also showed that oxidative stress reduced ARID1A gene expression., Competing Interests: DECLARATION OF CONFLICTING INTERESTS: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Disclosures and Ethics As a requirement of publication, author(s) have provided to the publisher signed confirmation of compliance with legal and ethical obligations including, but not limited to, the following: authorship and contributorship, conflicts of interest, privacy and confidentiality, and (where applicable) protection of human and animal research subjects. The authors have read and confirmed their agreement with the ICMJE authorship and conflict of interest criteria. The authors have also confirmed that this article is unique and not under consideration or published in any other publication, and that they have permission from rights holders to reproduce any copyrighted material. Any disclosures are made in this section. The external blind peer reviewers report no conflicts of interest.

Background: Cell culture techniques have many advantages for investigation of drug transport to target organ like liver. HepG2 and Huh-7 are two cell lines available from hepatoma that can be used as a model for hepatic drug transport. The present study is aimed to analyze the expression level of several drug transporter genes in two hepatoma cell lines, HepG2 and Huh-7 and their response to inhibitors., Materials and Methods: This is an in vitro study using HepG2 and Huh-7 cells. The expression level of the following drug transporter genes was quantified: P-glycoprotein/multidrug resistance protein 1, Organic Anionic Transporter Protein 1B1 (OATP1B1) and Organic Cationic Transporter-1 (OCT1). Ribonucleic acid was extracted from the cells using Tripure isolation reagent, then gene expression level of the transporters is quantified using Applied Biosystems quantitative reverse transcriptase polymerase chain reaction. Verapamil (P-glycoprotein inhibitor), nelfinavir (OATP1B1 inhibitor), quinidine (OCT1 inhibitor) were used to differentiate the inhibitory properties of these agents to the transporter expressions in HepG2 and Huh-7 cells., Results: Huh-7 shows a higher level of P-glycoprotein, OATP1B1 and OCT1 expressions compared with those of HepG2. Verapamil reduces the expressions of P-glycoprotein in HepG2 and Huh-7; nelfinavir reduces the expression of OATP1B1 in HepG2 and Huh-7; while quinidine reduces the OCT1 gene expressions in HepG2, but not in Huh-7 cells., Conclusion: This study indicates that HepG2 might be a more suitable in vitro model than Huh-7 to study drug transport in hepatocytes involving drug transporters.

Tamoxifen is the first line drug used in the treatment of estrogen receptor-positive (ER+) breast cancer. The development of multidrug resistance (MDR) to tamoxifen remains a major challenge in the treatment of cancer. One of the mechanisms related to MDR is decrease of drug influx via overexpression of drug efflux transporters such as P-glycoprotein (P-gp/MDR1), multidrug resistance associated protein (MRP), or BCRP (breast cancer resistance protein). We aimed to investigate whether the sensitivity of tamoxifen to the cells is maintained through the short period and whether the expressions of several drug efflux transporters have been upregulated. We exposed MCF7 breast cancer cells with tamoxifen 1 μM for 10 passages (MCF7 (T)). The result showed that MCF7 began to lose their sensitivity to tamoxifen from the second passage. MCF7 (T) also showed a significant increase in all transporters examined compared with MCF7 parent cells. The result also showed a significant increase of CC50 in MCF7 (T) compared to that in MCF7 (97.54 μM and 3.04 μM, resp.). In conclusion, we suggest that the expression of several drug efflux transporters such as P-glycoprotein, MRP2, and BCRP might be used and further studied as a marker in the development of tamoxifen resistance.

Chitosan, a multipurpose biomaterial, has been shown to exert effects against several types of cancer including oral cancer. However, the mechanisms underlying the anticancer activities of chitosan on oral squamous cell carcinoma (SCC) cells remain largely unknown. The present study aimed to compare the effects of low-molecular-weight chitosan (LMWC) and cisplatin on oral SCC Ca9-22 and non-cancer keratinocyte HaCaT cell lines. Cell viability and cell cycle profiles were measured by MTT assay and laser scanning cytometry, respectively. Apoptosis was examined by TUNEL assay and electron microscopy, followed by analysis of caspase activity. LMWC exhibited cytotoxic effects on Ca9-22, but not HaCaT cells, whereas cisplatin induced apoptosis in both types of cells. Exposure of Ca9-22 cells to LMWC led to G1/S cell cycle arrest and an increase of TUNEL-positive cells accompanied by an early apoptotic cell morphology and subtle increases of caspase activity. Short-term LMWC exposure was less cytotoxic to HaCaT cells than to Ca9-22 cells, and anticancer activity was exerted through induction of apoptosis and cell cycle arrest, suggesting that LMWC could be a promising natural anticancer agent with fewer side effects on normal cells.

We investigated whether in addition to the well known genetic alteration in red blood cell membrane band 3 protein, a deletion of 9 amino acids leading to ovalocytosis, other mutations to band 3 also exist. In 12% of our thalassemia major patients investigated, we found two bands in the agarose gel-electrophoresis of PCR products from band 3 gene with a difference of 65 +/- 10 bp, equivalent to a deletion of 20 to 25 amino acids in band 3 protein. Thus, a co-existing band 3-mutant allele in addition to the thalassemic globin gene defects, could also contribute to erythrocyte membrane defects and to the spectrum of clinical symptoms of these thalassemia major patients.

Aim: to analyze the role of allopurinol in reducing the ischemia-reperfusion injury, and to confirm the HIF-1 binding activity changes during the surgical correction of tetralogy of fallot (TF)., Methods: randomized, double-blind experimental study on patients undergoing surgical correction of TF in the Integrated Cardiovascular Services, Cipto Mangunkusumo Hospital from September 2009-May 2010. Patients were randomly divided into two groups. The first group was given 10 mg/kg body weight of allopurinol 3 times before undergoing operation (n=13) and the other group was given placebo (n=13). Tissue specimen from right ventricular muscle were taken for measurement of reactive oxygen species (ROS) expression and blood specimens from intra-coronary sinus for measurement of TNF-, superoxide dismutase (SOD), and malondialdehyde (MDA)., Results: cardiomyocytes expressing ROS in placebo group increased (41.37±29.29%; 42.61±22.82% and 53.81±25.76%), while in allopurinol group decreased (44.68±19.79%, 56.87±15.50%, and 47.98±22.52%). Concentration of TNF- tend to decrease in allopurinol group, while it tended to increase in the placebo goup. Concentration of SOD increased in the allopurinol group, while in the placebo group there were no significant changes. Concentration of MDA was highly increased in the placebo group (1.75 pmol/mg; 2.37 pmol/mg; 2.72 pmol/mg; 4.82 pmol/mg), but statistically it was not significant. On the other hand, there was no significant change of MDA concentration in the allopurinol group. Expression of HIF-1 in TF patients decreased significantly (p=0.026) from pre ischemic phase to ischemic phase, but it increased in the reperfusion phase (0.95 OD/mg protein, 0.52 OD/mg protein, 0.9 OD/mg protein)., Conclusion: pre-surgical oral allopurinol treatment reduced ischemia-reperfusion injury during TF surgical correction, as indicated by the decrease in the number of cardiomyocytes expressing ROS, reducing TNF-, SOD, and MDA concentration in blood. There was a decrease in HIF-1 concentration in cardiomyocytes of the right ventricle during ischemic phase.

Aim: to assess the effect of systemic hypoxia on gastric mucosa and the activation of stress-responsive transcription factors induced by hypoxia., Methods: in this experimental study, rats were allocated to control and experimental groups. The experimental group was divided into subgroups and subjected to hypoxia conditions for 1, 7, 14 or 21 days. Afterwards, histopathological evaluation and study of the protein expression of the gastric mucosa were performed., Results: the results showed that longer exposure to hypoxic conditions leads to more severe gastric ulceration. Twenty-four hours after induction, 60% of rats had developed gastric ulcers. Seven days after induction, 80% of rats developed gastric ulcers. In the 14-day and 21-day hypoxia conditions, epithelialization (a sign of gastric ulcer healing) was observed. Evaluation of the average ulcer depth on the day of treatment showed that the greatest depth was on day 7, and the shallowest was on day 21 of treatment. Western blot analyses demonstrated that systemic hypoxia resulted in the expression of heat shock factor (HSF) and heat shock protein 70 (HSP-70), which were highest on day 7 and then regressed gradually. In control, HSF-1 and HSP-70 were not detected by Western blot analysis in the control group (normoxia)., Conclusion: in this study, systemic hypoxia caused gastric ulcers, and during the time of exposure to hypoxia, an adaptation process in the form of gastric epithelialization occurred in the rats. This development of gastric lesions was in line with the expression pattern of HSF-1 HIF-1 and HSP-70.

Lung cancer is the primary cause of cancer death in the world. Although it is well established that tobacco smoke causes lung cancer, not all smokers develop lung cancer. Manganese superoxide dismutase (MnSOD), a major determinant of antioxidants in matrix mitochondria, plays a pivotal role in eliminating anion superoxide free radical generated from the tobacco smoke. The aim of this study was to analyze the enzyme activity of MnSOD in blood of lung cancer patients with a smoking history in relationship to oxidative stress. Samples were taken from leukocyte cells of 20 lung cancer patients in Persahabatan Hospital Jakarta. Control groups included 50 healthy smokers and 50 non smokers, all aged over 40 years. The MnSOD activity determined biochemically based on the inhibition of xanthin oxidase, of lung cancer patients was lower than the control group's (p<0.001). Plasma MDA levels, determined by reaction with thiobarbituric acid (TBA), were not significantly different (p=0.479), whereas plasma carbonyl levels were elevated (p=0.003). Free radical production in lung cancer patients thus appeared high. Smoker controls also tended to exhibit lower MnSOD and higher carbonyl radicals than their non-smoking counterparts. Continue cigarette smoke exposure may increase production of ROS and bring about a reduction of MnSOD, which could play a role in lung cancer development.

Aim: to explore the effects of chronic systemic hypoxia on myocardial structure and morphology. In addition, the goal of present study is to develop a hypoxia-induced heart failure model in rats., Methods: Sprague-Dawley male rats, weighing 220-250 g at the time of recruitment were randomly allocated into 7 groups (n = 4 per group), the control normoxia group was exposed to room air, while the hypoxia groups were caged in a plexiglas hypoxic chamber (8% O2 and 92% N2) for 28 days. Structural and morphological changes of ventricular myocardium were determined at day 28, while blood gas parameters were measured at day 1, 3, 7, 14, 21, and 28., Results: histopathologic and morphologic evaluation showed massive hypertrophy accompanied by damage of the intercalated disk (ID) structure, angiogenesis, necrosis, fibrosis, and apoptosis as a hallmark of ventricular remodeling. At the end of treatment, there were increases of LV (2.79 vs 3.71) and RV (1.72 vs 2.54) wall thicknesses, and also in hypertrophy index (from 3.19 to 5.74). Blood gas analysis revealed metabolic acidosis compensated by respiratory alkalosis. There was an observed decrease of blood gas parameters in hypoxia group compared to control group: PO2 (24.7 vs 96.4 mm Hg), PCO2 (18.2 vs 40.4 mm Hg), O2 saturation (25.5 vs 94.1 %), and HCO3 (10.1 vs 23.4 mmol/L). On the other hand an increase in hemoglobin level (221.5 vs 120.3 g/L), haematocrit level (68.6 vs 45.2 %), and red blood cell count (10.4 vs 6.9 μL/1000) could be observed., Conclusion: our data clearly show that chronic systemic hypoxia causes massive ventricular hypertrophy accompanied by severe structural and morphological impairment of ventricular myocardium, which eventually results in cardiac failure.

Aim: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and its relation with oxidative stress in liver of rats induced by systemic chronic normobaric hypoxia., Methods: Twenty five male, 6-8 weeks old rats were induced by systemic hypoxia. Rats were divided randomly into 5 groups (n = 5 per group). The control group was exposed to normal environment while the hypoxic groups were kept in hypoxic chamber (10% O(2)) for 1, 3, 7, and 14 days. Animals were sacrificed, the liver isolated and homogenized. Total RNA was extracted and isolated and expression of HIF-1alpha mRNA was measured by real-time RT PCR using Pffafl method. Malondialdehyde (MDA), product of lipid peroxidation was measured by tBARS assay. Glutathione (GSH), an abundant endogenous antioxidant in the liver tissue was measured using Ellman method., Results: Study showed that expression of HIF-1alpha mRNA was increased in group treated for 1 day of hypoxic condition, and then decreased in group treated for 3, 7 and 14 days of hypoxic condition related with duration of hypoxic condition. The MDA level in liver tissue increased, but not significant in all groups of hypoxic condition and persisted along duration time of hypoxic condition. The GSH level was decreased significantly (p<0.005) in all groups of hypoxic condition., Conclusion: Expression of HIF-1alpha mRNA was higher at the early phase of hypoxia and decreased as hypoxia continued. Systemic hypoxia induction caused increased ROS formation during hypoxia, and depleted the GSH concentration in the liver. Oxidative stress present in liver of rat was induced by systemic hypoxia.

Aim: To investigate the expression of B-type natriuretic peptide-45 (BNP-45) gene which was induced by systemic chronic hypoxia, and whether these changes would be different from BNP-45 protein in the plasma and its mRNA in the ventricular myocardial. This study also aimed to test the hypothesis that systemic chronic hypoxia may cause heart failure., Methods: Although clinical use of BNP as a biomarker in heart failure is increasing, the specificity of BNP for heart failure is not robust, suggesting that other mechanisms beyond simple ventricular stretch stimulate BNP release. Plasma BNP levels were markedly increased in patients with coronary artery disease but without concomitant left ventricular dysfunction. Thus, elevated BNP levels do not necessarily reflect heart failure but may also result from cardiac ischemia. Sprague-Dawley male rats, weighing 220-250 g at the time of recruitment were randomly divided into 7 groups (n = 4 per group), the control normoxia group was exposed to room air, while the hypoxia group were caged in a plexiglass hypoxic chamber (8%O2 and 92% N2) for 1, 3, 7, 14, 21, and 28 days, respectively., Results: Our data clearly showed that plasma BNP-45 and ventricular BNP-45 mRNA concentration were markedly increased which reached its peak on day 21 after treatment., Conclusion: Regulation of BNP-45 gene expression occurred at transcription as well as post-transcription level. Systemic chronic hypoxia could result in heart failure, especially when the hypoxia is severe and prolonged.

Ulcer healing process is an intricate and active process including reconstruction process of mucosa through formation of granulation tissue. Granulation tissue formation takes place by means of formation of ulcer base, formation of blood vessel (angiogenesis) and re-establishment of glandular architecture. The process of granulation tissue formation on the ulcer base takes place 48-72 hours after ulceration process occurs. These three phases involve various genes grouped according to their activated time, i.e. the initial response genes, intermediate response gene and late response genes. Initial response genes are activated in 30 minutes to 2 hours time, e.g EGF-R, c-fos, c-jun, egr-1, Sp-1, TFF-2/SP. Intermediate response genes are activated for 6 hours to 2 days, eg EGF, bFGF, PDGF and VEGF. Late response genes are activated for 14 days, e.g. HGF, ITF, c-met/HGF-R.