Estrogen Receptor Is Required for Metformin-Induced Apoptosis in Breast Cancer Cells Under Hyperglycemic Conditions.

Backgrounds: About 25% to 30% of estrogen receptor (ER)-positive breast cancer patients develop resistance to endocrine therapy. Human epidermal growth factor receptor 2 (HER2) has been shown to cooperate with several growth factors that regulate cellular energy metabolism, including the insulin-like growth factor 1 receptor (IGF-1R). Objective: As the first-line therapy for type 2 diabetes mellitus (T2DM) patients, metformin is widely known to inhibit the metabolic reprogramming of cancer cells. This study aims to investigate metformin's efficacy in inhibiting endocrine resistance related to genes regulating energy metabolism in both ER-positive and ER-negative breast cancer cell lines under hyperglycemic conditions. Design and methods: MDA-MB-361 (ER-positive, HER2-positive) and SKBR3 (ER-negative, HER2-positive) cancer cell lines were used to represent ER status. Cell viability and cell survival rate were measured using the colorimetric assay of Cell Counting Kit-8. All mRNA levels were quantified using real-time quantitative polymerase chain reaction preceded by reverse transcription. A P value of <.05 was considered statistically significant. Results: Unlike MDA-MB-361, SKBR3 were found to acquire resistance upon metformin treatment in hyperglycemic conditions. Moreover, the mRNA expression of IGF-1R and its downstream signaling, such as the mammalian target of rapamycin (mTOR), was not affected by metformin. Meanwhile, the mRNA expression level of ribosomal S6 kinase 1 (S6K1) was upregulated, whereas forkhead box O1 (FOXO1) was downregulated after metformin treatment in hyperglycemic conditions. Conclusions: This preliminary study suggests that an alternative pathway of metformin resistance may exist in the absence of ERα. Therefore, relying solely on metformin may be inadequate to inhibit the aggressiveness of breast cancer cells.

Navigating metformin's impact on breast cancer: insights into resistance, alternative pathways, and the crucial role of estrogen receptor under high-glucose conditions Around 25% to 30% of breast cancer patients with estrogen receptor (ER)-positive tumors become resistant to hormone therapy. This study explores whether metformin, a drug commonly used for type 2 diabetes, can counteract this resistance by affecting genes linked to energy metabolism. The research focused on both ER-positive (MDA-MB-361) and ER-negative (SKBR3) breast cancer cell lines under high-glucose conditions. Results showed that although metformin inhibited the growth of ER-positive cells, it surprisingly promoted resistance in ER-negative cells. The expression of insulin-like growth factor 1 receptor (IGF-1R) and its downstream signals like mammalian target of rapamycin (mTOR) remained unaffected by metformin. However, metformin did alter the expression of other genes related to energy metabolism, suggesting that a different resistance pathway might exist in ER-negative cases. In conclusion, this early study implies that relying solely on metformin might not be sufficient to combat the aggressiveness of breast cancer cells, particularly in cases lacking ERα. More research is needed to understand alternative pathways and develop more effective strategies against resistance.

Development and validation of cost-effective SYBR Green-based RT-qPCR and its evaluation in a sample pooling strategy for detecting SARS-CoV-2 infection in the Indonesian setting.

A low-cost SYBR Green-based RT-qPCR method to detect SARS-CoV-2 were developed and validated. Primers targeting a conserved and vital region of the N genes of SARS-CoV-2 were designed. In-silico study was performed to analyse the compatibility of the selected primer pair with Indonesian SARS-CoV-2 genome sequences available from the GISAID database. We determined the linearity of our new assay using serial dilution of SARS-CoV-2 RNA from clinical samples with known virus concentration. The assay was then evaluated using clinically relevant samples in comparison to a commercial TaqMan-based test kit. Finally, we applied the assay in sample pooling strategies for SARS-CoV-2 detection. The SYBR Green-based RT-qPCR method was successfully developed with sufficient sensitivity. There is a very low prevalence of genome variation in the selected N primer binding regions, indicating their high conservation. The validation of the assay using clinical samples demonstrated similar performance to the TaqMan method suggesting the SYBR methods is reliable. The pooling strategy by combining 5 RNA samples for SARS-CoV-2 detection using the SYBR RT-qPCR methods is feasible and provides a high diagnostic yield. However, when dealing with samples having a very low viral load, it may increase the risk of missing positive cases.

Low-cost SYBR Green-based RT-qPCR assay for detecting SARS-CoV-2 in an Indonesian setting using WHO-recommended primers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for the ongoing coronavirus disease 2019 (COVID-19) pandemic. For laboratory diagnosis, low-cost detection of SARS-CoV-2 is urgently needed, particularly in developing countries with limited resources. Probe- or TaqMan-based real-time reverse transcription polymerase chain reaction (RT-qPCR) is currently the gold standard for diagnosing infected individuals, as recommended by the World Health Organization (WHO). However, this assay is expensive, making it difficult to use for diagnosis on a large scale. Therefore, in this study, we develop and validate an alternative approach for RT-qPCR diagnosis by employing the DNA intercalating dye SYBR Green. We evaluate and use two WHO-recommended primers, namely CCDC-N and HKU-ORF1b-nsp14. The compatibility of the two primers was tested in silico with Indonesian SARS-CoV-2 genome sequences retrieved from the GISAID database and using bioinformatic tools. Using in vitro-transcribed RNA, optimization, sensitivity, and linearity of the two assays targeting the N and Nsp-14 genes were carried out. For further evaluation, we used clinical samples from patients and performed the SYBR Green-based RT-qPCR assay protocol in parallel with TaqMan-based commercial assay. Our results show that our methodology performs similarly to the broadly used TaqMan-based detection method in terms of specificity and sensitivity and thus offers an alternative assay for the detection of SARS-CoV-2 RNA for diagnostic purposes.

Synthesis conditions and characterization of superparamagnetic iron oxide nanoparticles with oleic acid stabilizer.

Superparamagnetic iron oxide nanoparticles (SPIONs), part of magnetic nanoparticles, have been widely used in biomedical applications. Biocompatibility and magnetic properties make the SPIONs developed further by a lot of researchers. However, in the synthesis process, SPIONs can run into agglomeration. Oleic acid (OA) is one of the stabilizers to prevent agglomeration. This research aims to optimize the synthesis conditions and characterization of SPIONs with OA as a stabilizer. The synthesis of Superparamagnetic Iron Oxide Nanoparticles-Oleic Acid (SPIONs-OA) was performed using the coprecipitation method and was prepared with the addition of 0.75, 1.5, and 3%v/v OA and stirring rate of 750, 1500, 3000, 6000, 9000, and 12,000 rpm. The characterization of hydrodynamic size and polydispersity index was evaluated by dynamic light scattering. Meanwhile, the crystal structure was observed by X-ray diffraction. Then, Fourier transform infrared spectroscopy (FTIR) was used to analyze structures. The results showed that the hydrodynamic size was dependent on OA concentrations and stirring rate. The addition of 1.5%v/v OA and stirring conditions of 750 rpm resulted in the smallest hydrodynamic size and polydispersity index (83.71 ± 0.70 nm and 0.215 ± 0.01 nm, respectively). Based on the crystal structure analysis, the crystal shape was magnetic cubic, and the size of Fe3O4 crystallite changed from 11.38 to 5.61 nm. The FTIR indicated a strong chemical bond between the hydroxyl group of SPIONs and carboxylic acid of OA. In conclusion, the SPIONs-OA was successfully prepared with 1.5%v/v OA concentrations and a stirring rate of 750 rpm.

In vitro evaluation of synergistic inhibitory effects of neuraminidase inhibitors and methylglyoxal against influenza virus infection.

BACKGROUND AND AIMS: Influenza virus infections are serious public health concerns worldwide that cause considerable mortality and morbidity. Moreover, the emergence of resistance to anti-influenza viral agents underscores the need to develop new anti-influenza viral agents and novel treatment strategies. Recently, we identified anti-influenza viral activity of manuka honey. Therefore, we hypothesized that methylglyoxal (MGO), a key component of manuka honey, may impart anti-influenza viral activity. The aim of this study was to evaluate the anti-influenza viral activity of MGO and its potential in combination treatments with neuraminidase (NA) inhibitors. METHODS: MDCK cells were used to evaluate anti-influenza viral activity. To evaluate the mechanism of MGO, plaque inhibition assays were performed. The synergistic effects of MGO and viral NA inhibitors were tested. RESULTS: MGO inhibited influenza virus A/WSN/33 replication 50% inhibitory concentration = 240 ± 190 µM; 50% cytotoxic concentration = 1.4 ± 0.4 mM; selective index (SI) = 5.8, which is related to its virucidal effects. Moreover, we found that MGO showed promising activity against various influenza strains. A synergistic effect was observed by a marked increase in SI of NA inhibitors at ∼1/100(th) of their single usage. A synergistic effect of MGO and oseltamivir was also observed against oseltamivir-resistant virus. CONCLUSIONS: Our results showed that MGO has potent inhibitory activity against influenza viruses and also enhanced the effect of NA inhibitors. Thus, the co-administration of MGO and NA inhibitors should be considered for treatment of influenza virus infections.

Anti-influenza viral effects of honey in vitro: potent high activity of manuka honey.

BACKGROUND AND AIMS: Influenza viruses are a serious threat to human health and cause thousands of deaths annually. Thus, there is an urgent requirement for the development of novel anti-influenza virus drugs. Therefore, the aim of this study was to evaluate the anti-influenza viral activity of honey from various sources. METHODS: Antiviral activities of honey samples were evaluated using MDCK cells. To elucidate the possible mechanism of action of honey, plaque inhibition assays were used. Synergistic effects of honey with known anti-influenza virus drugs such as zanamivir or oseltamivir were tested. RESULTS: Manuka honey efficiently inhibited influenza virus replication (IC50 = 3.6 ± 1.2 mg/mL; CC50 = 82.3 ± 2.2 mg/mL; selective index = 22.9), which is related to its virucidal effects. In the presence of 3.13 mg/mL manuka honey, the IC50 of zanamivir or oseltamivir was reduced to nearly 1/1000th of their single use. CONCLUSIONS: Our results showed that honey, in general, and particularly manuka honey, has potent inhibitory activity against the influenza virus, demonstrating a potential medicinal value.