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Isolation and Characterization of Cancer Stem Cells of the Non-Small-Cell Lung Cancer (A549) Cell Line

Halim NHA, Zakaria N, Satar NA, Yahaya BH

Methods Mol Biol, 2016;1516:371-388.
PMID: 27032945 DOI: 10.1007/7651_2016_326

Cancer is a major health problem worldwide. The failure of current treatments to completely eradicate cancer cells often leads to cancer recurrence and dissemination. Studies have suggested that tumor growth and spread are driven by a minority of cancer cells that exhibit characteristics similar to those of normal stem cells, thus these cells are called cancer stem cells (CSCs). CSCs are believed to play an important role in initiating and promoting cancer. CSCs are resistant to currently available cancer therapies, and understanding the mechanisms that control the growth of CSCs might have great implications for cancer therapy. Cancer cells are consist of heterogeneous population of cells, thus methods of identification, isolation, and characterisation of CSCs are fundamental to obtain a pure CSC populations. Therefore, this chapter describes in detail a method for isolating and characterizing a pure population of CSCs from heterogeneous population of cancer cells and CSCs based on specific cell surface markers.
Exercise prevents leptin-induced increase in blood pressure in Sprague-Dawley rats

Farhana K, Effendi I, Caszo B, Satar NA, Singh HJ

J Physiol Biochem, 2014 Jun;70(2):417-23.
PMID: 24711061 DOI: 10.1007/s13105-014-0319-2

Although leptin has been shown to increase blood pressure (BP), it is however unclear if this increase can be prevented by exercise. This study therefore investigated the effect of leptin treatment with concurrent exercise on blood pressure (BP), sodium output, and endothelin-1 (ET-1) levels in normotensive rats. Male Sprague-Dawley rats weighing 250-270 g were divided into four groups consisting of a control group (n = 6), leptin-treated (n = 8), non-leptin-treated exercise group (n = 8), and a leptin-treated exercise group (n = 8). Leptin was given subcutaneously daily for 14 days (60 μg/kg/day). Animals were exercised on a treadmill for 30 min at a speed of 0.5 m/s and at 5° incline four times per week. Measurement of systolic blood pressure (SBP) and collection of urine samples for estimation of sodium and creatinine was done once a week. Serum samples were collected at the end of the experiment for determination of sodium, creatinine and ET-1. At day 14, mean SBP and serum ET-1 level in the leptin-treated group was significantly higher than that in the control group whereas mean SBP and serum ET-1 level was significantly lower in the leptin-treated exercise group than those in leptin-treated and control groups. Creatinine clearance, urinary sodium excretion, and urine output were not different between the four groups. Regular treadmill exercise prevents leptin-induced increases in SBP in rats, which might in part result from increased urinary sodium excretion and preventing the leptin-induced increases in serum ET-1 concentration.
Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo

Jusof WH, Khan NA, Rajikin MH, Satar NA, Mustafa MF, Jusoh N, et al.

Int J Fertil Steril, 2015 07 27;9(2):221-9.
PMID: 26246881

BACKGROUND: Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos.
MATERIALS AND METHODS: In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test.
RESULTS: A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001).
CONCLUSION: Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification.
Fulltext Targeting Lung Cancer Stem Cells: Research and Clinical Impacts

Zakaria N, Satar NA, Abu Halim NH, Ngalim SH, Yusoff NM, Lin J, et al.

Front Oncol, 2017;7:80.
PMID: 28529925 DOI: 10.3389/fonc.2017.00080

Lung cancer is the most common cancer worldwide, accounting for 1.8 million new cases and 1.6 million deaths in 2012. Non-small cell lung cancer (NSCLC), which is one of two types of lung cancer, accounts for 85-90% of all lung cancers. Despite advances in therapy, lung cancer still remains a leading cause of death. Cancer relapse and dissemination after treatment indicates the existence of a niche of cancer cells that are not fully eradicated by current therapies. These chemoresistant populations of cancer cells are called cancer stem cells (CSCs) because they possess the self-renewal and differentiation capabilities similar to those of normal stem cells. Targeting the niche of CSCs in combination with chemotherapy might provide a promising strategy to eradicate these cells. Thus, understanding the characteristics of CSCs has become a focus of studies of NSCLC therapies.
Standardized ethanol-water extract of Ficus deltoidea Angustifolia reduces blood pressure in spontaneously hypertensive rats

Kamal MSA, Ismail NH, Satar NA, Azis NA, Radjeni Z, Mohammad Noor HS, et al.

Clin Exp Hypertens, 2019;41(5):444-451.
PMID: 30648895 DOI: 10.1080/10641963.2018.1506467

Ficus deltoidea is used in Malay traditional medicine for the treatment of a number of disorders, including hypertension. There is, however, no scientific evidence on its anti-hypertensive effects. This study, therefore, investigated the effects of a standardized ethanolic-water extract of Ficus deltoidea Angustifolia (FD-A) on blood pressure (BP) in spontaneously hypertensive rats (SHR). Male SHR with systolic BP of >150 were divided into 4 groups (n = 8) and given either FD-A (800 or 1000 mg kg-1 day-1) or losartan (10 mg kg-1 day-1) or 0.5 ml of distilled water (control) daily for 28 days. BP, body weight, food and water intake, serum and urinary electrolytes, endothelin-1 (ET-1), total antioxidant capacity (TAC) and components of the renin-angiotensin-aldosterone system were measured. Data were analyzed using ANOVA with statistical significance set at p
Blood pressure and urine metabolite changes in spontaneously hypertensive rats treated with leaf extract of Ficus deltoidea var angustifolia

Kamal MSA, Mediani A, Kasim N, Ismail NH, Satar NA, Azis NA, et al.

J Pharm Biomed Anal, 2022 Feb 20;210:114579.
PMID: 35016031 DOI: 10.1016/j.jpba.2021.114579

Ficus deltoidea var angustifolia (FD-A) reduces blood pressure in spontaneously hypertensive rats (SHR) but the mechanism remains unknown. Changes in urine metabolites following FD-A treatment in SHR were, therefore, examined to identify the mechanism of its antihypertensive action. Male SHR were given either FD-A (1000 mg kg-1 day-1) or losartan (10 mg kg-1 day-1) or 0.5 mL of ethanolic-water (control) daily for 4 weeks. Systolic blood pressure (SBP) was measured every week and urine spectra data acquisition, on urine collected after four weeks of treatment, were compared using multivariate data analysis. SBP in FD-A and losartan treated rats was significantly lower than that in the controls after four weeks of treatment. Urine spectra analysis revealed 24 potential biomarkers with variable importance projections (VIP) above 0.5. These included creatine, hippurate, benzoate, trimethylamine N-oxide, taurine, dimethylamine, homocysteine, allantoin, methylamine, n-phenylacetylglycine, guanidinoacetate, creatinine, lactate, glucarate, kynurenine, ethanolamine, betaine, 3-hydroxybutyrate, glycine, lysine, glutamine, 2-hydroxyphenylacetate, 3-indoxylsulfate and sarcosine. From the profile of these metabolites, it seems that FD-A affects urinary levels of metabolites like taurine, hypotaurine, glycine, serine, threonine, alanine, aspartate and glutamine. Alterations in these and the pathways involved in their metabolism might underlie the molecular mechanism of its antihypertensive action.
Fulltext Novel triple‑positive markers identified in human non‑small cell lung cancer cell line with chemotherapy-resistant and putative cancer stem cell characteristics

Satar NA, Fakiruddin KS, Lim MN, Mok PL, Zakaria N, Fakharuzi NA, et al.

Oncol Rep, 2018 Aug;40(2):669-681.
PMID: 29845263 DOI: 10.3892/or.2018.6461

Through the specific identification and direct targeting of cancer stem cells (CSCs), it is believed that a better treatment efficacy of cancer may be achieved. Hence, the present study aimed to identify a CSC subpopulation from adenocarcinoma cells (A549) as a model of non‑small cell lung cancer (NSCLC). Ιnitially, we sorted two subpopulations known as the triple‑positive (EpCAM+/CD166+/CD44+) and triple‑negative (EpCAM-/CD166-/CD44-) subpopulation using fluorescence-activated cell sorting (FACS). Sorted cells were subsequently evaluated for proliferation and chemotherapy-resistance using a viability assay and were further characterized for their clonal heterogeneity, self-renewal characteristics, cellular migration, alkaline dehydrogenase (ALDH) activity and the expression of stemness-related genes. According to our findings the triple‑positive subpopulation revealed significantly higher (P<0.01) proliferation activity, exhibited better clonogenicity, was mostly comprised of holoclones and had markedly bigger (P<0.001) spheroid formation indicating a better self-renewal capacity. A relatively higher resistance to both 5‑fluouracil and cisplatin with 80% expression of ALDH was observed in the triple‑positive subpopulation, compared to only 67% detected in the triple‑negative subpopulation indicated that high ALDH activity contributed to greater chemotherapy-resistance characteristics. Higher percentage of migrated cells was observed in the triple‑positive subpopulation with 56% cellular migration being detected, compared to only 19% in the triple‑negative subpopulation on day 2. This was similarly observed on day 3 in the triple‑positive subpopulation with 36% higher cellular migration compared to the triple‑negative subpopulation. Consistently, elevated levels of the stem cell genes such as REX1 and SSEA4 were also found in the triple‑positive subpopulation indicating that the subpopulation displayed a strong characteristic of pluripotency. In conclusion, our study revealed that the triple‑positive subpopulation demonstrated similar characteristics to CSCs compared to the triple‑negative subpopulation. It also confirmed the feasibility of using the triple‑positive (EpCAM+/CD166+/CD44+) marker as a novel candidate marker that may lead to the development of novel therapies targeting CSCs of NSCLC.