Septelia Inawati Wanandi

Drug Efflux Transporters Are Overexpressed in Short-Term Tamoxifen-Induced MCF7 Breast Cancer Cells

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.

Differential expression of several drug transporter genes in HepG2 and Huh-7 cell lines

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.

Comparison of Effective α-Tocopherol Concentrations Ameliorating Reduced Glucose Transport and Counteracting t-Butyl hydroxyperoxide- Induced Lipid Peroxidation in Isolated Erythrocytes from β-Thalassemic Patients

In thalassemia patients Red Blood Cell (RBC) membrane is the major target of oxidative stress caused by hemichrome radicals. Moreover, RBC membranes from thalassemic patients are more susceptible to oxidative stress, which causes lipid peroxidation, membrane rigidity, and dysfunction of membrane proteins. We investigated hemoglobin-free isolated RBC membranes (ghosts) from 28 β-thalassemia major patients and from 24 normal blood samples. Our research aimed to determine the effective concentrations of racemic α-tocopherol i) counteracting experimental oxidative stress induced by 2mM tertiary butylhydroperoxide (t-BHP) and ii) ameliorating impaired glucose transport in RBC ghosts from thalassemia patients. Reduced glucose transport in thalassemic ghosts was ameliorated significantly by tocopherol with a maximum effect at 75 ppm concentration. Furthermore, ghosts were pre-incubated with/without α-tocopherol up to 200 ppm before treated with t-BHP as the oxidative agent and reactive membrane thiols determined. As a parameter of lipid peroxidation (LPO) thiobarbiturate-reactive substances were measured and expressed as malondialdehyde levels. Tocopherol counteracted LPO continuously in a concentration-dependent way up 200 ppm. Hence, it is concluded that tocopherol acts at different concentrations as an antioxidant and on glucose transport by GLUT1 in RBC membranes.

Metabolic Interplay between Tumour Cells and Cancer-Associated Fibroblasts (CAFs) under Hypoxia versus Normoxia

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.

Neuroglobin correlates with cryptochrome-1 in obstructive sleep apnea with primary aldosteronism

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

Glucosamine decreases the stemness of human ALDH+ breast cancer stem cells by inactivating STAT3

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.

Curcumin for the Prevention of Epithelial-Mesenchymal Transition in Endoxifen-Treated MCF-7 Breast Cancer Cells

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+beta-estradiol 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.