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Fulltext The Cytotoxic Properties of Baeckea frutescens Branches Extracts in Eliminating Breast Cancer Cells

Shahruzaman SH, Mustafa MF, Ramli S, Maniam S, Fakurazi S, Maniam S

Evid Based Complement Alternat Med, 2019;2019:9607590.
PMID: 31178918 DOI: 10.1155/2019/9607590

Breast cancer is the leading cause of cancer death in women in over 100 countries worldwide and accounts for almost 1 in 4 cancer cases among women. Baeckea frutescens of the family Myrtaceae has been used in traditional medicine and is known to possess antibacterial, antipyretic, and cytoprotective properties. In this study, we investigated the role of Baeckea frutescens branches extracts against human breast cancer cells. Baeckea frutescens branches extracts were prepared using Soxhlet apparatus with solvents of different polarity. The selective cytotoxic activity and the glucose consumption rate of Baeckea frutescens branches extracts of various concentrations (20 to 160 ug/ml) at 24-, 48-, and 72-hour time points were studied using MTT and glucose uptake assay. The IC50 values in human breast cancer (MCF-7 and MDA-MB-231) and mammary breast (MCF10A) cell lines were determined. Apoptotic study using AO/PI double staining was performed using fluorescent microscopy. The glucose uptake was measured using 2-NBDG, a fluorescent glucose analogue. The phytochemical screening of major secondary metabolites in plants was performed. This study reports that Baeckea frutescens branches extracts showed potent selective cytotoxic activity against MCF-7 cells compared to MDA-MB-231 cells after 72 hours of treatment. Evidence of early apoptosis which includes membrane blebbing and chromatin condensation was observed after 72 hours of treatment with Baeckea frutescens branches extracts. Interestingly, for the glucose uptake assay, the inhibition was observed as early as 24 hours upon treatment. All Baeckea frutescens extracts showed the presence of major secondary metabolites such as tannin, triterpenoid, flavonoid, and phenol. However, alkaloid level was unable to be determined. The identification of Baeckea frutescens and its possible role in selectively inhibiting glucose consumption in breast cancer cells defines a new role of natural product that can be utilised as an effective agent that regulates metabolic reprogramming in breast cancer.
Fulltext The cytotoxic effect and glucose uptake modulation of Baeckea frutescens on breast cancer cells

Shahruzaman SH, Mustafa MF, Ramli S, Maniam S, Fakurazi S, Maniam S

BMC Complement Altern Med, 2019 Aug 19;19(1):220.
PMID: 31426778 DOI: 10.1186/s12906-019-2628-z

BACKGROUND: Baeckea frutescens (B. frutescens) of the family Myrtaceae is a plant that has been used in traditional medicine. It is known to have antibacterial, antipyretic and cytoprotective properties. The objective of this study is to explore the mechanism of B. frutescens leaves extracts in eliminating breast cancer cells.
METHOD: B. frutescens leaves extracts were prepared using Soxhlet apparatus with solvents of different polarity. The selective cytotoxicity of these extracts at various concentrations (20 to 160 μg/ml) were tested using cell viability assay after 24, 48 and 72 h of treatment. The IC50 value in human breast cancer (MCF-7 and MDA-MB-231) and mammary breast (MCF10A) cell lines were determined. Apoptotic study using AO/PI double staining was performed using fluorescent microscope. The glucose uptake was measured using 2-NBDG, a fluorescent glucose analogue. The phytochemical screening was performed for alkaloids, flavonoids, tannins, triterpenoids, and phenols.
RESULTS: B. frutescens leaves extracts showed IC50 value ranging from 10 -127μg/ml in MCF-7 cells after 72 h of treatment. Hexane extract had the lowest IC50 value (10μg/ml), indicating its potent selective cytotoxic activity. Morphology of MCF-7 cells after treatment with B. frutescens extracts exhibited evidence of apoptosis that included membrane blebbing and chromatin condensation. In the glucose uptake assay, B. frutescens extracts suppressed glucose uptake in cancer cells as early as 24 h upon treatment. The inhibition was significantly lower compared to the positive control WZB117 at their respective IC50 value after 72 h incubation. It was also shown that the glucose inhibition is selective towards cancer cells compared to normal cells. The phytochemical analysis of the extract using hexane as the solvent in particular gave similar quantities of tannin, triterpenoids, flavonoid and phenols. Presumably, these metabolites have a synergistic effect in the in vitro testing, producing the potent IC50 value and subsequently cell death.
CONCLUSION: This study reports the potent selective cytotoxic effect of B. frutescens leaves hexane extract against MCF-7 cancer cells. B. frutescens extracts selectively suppressed cancer cells glucose uptake and subsequently induced cancer cell death. These findings suggest a new role of B. frutescens in cancer cell metabolism.
Fulltext The immediate outcome of ventilation for pulmonary diseases

Yaacob I, Mustafa M

Singapore Med J, 1994 Oct;35(5):512-4.
PMID: 7701374

Fifty-eight patients were ventilated for acute respiratory failure complicating respiratory diseases between 1985 to 1990. There were 19 cases of chronic obstructive airway disease (COAD), 17 cases of asthma, 16 cases of pneumonia and 6 cases with other diagnoses. Overall, 40% of patients survived and were discharged from the ward. Patients with pneumonia had the lowest survival rate (25%) whilst the survival rates for asthma and COAD were 47% and 42% respectively. Increasing age had an inverse relationship to survival rate (r = 0.96, p < 0.05) but the duration of ventilation did not correlate with survival. Patients who were electively ventilated before respiratory arrest had a better chance of survival (57%) compared with only 18% survival rate in patients who were ventilated as an emergency (x2 = 4.47, p < 0.05). Patients who developed other organ failure had higher mortality (71%) than those who did not (22%; x2 = 2.14, p < 0.05). We conclude that patients younger than 50 years of age, who were electively ventilated and without other organ failure had a better immediate survival after assisted ventilation.
Fulltext The role of miRNAs 34a, 146a, 320a and 542 in the synergistic anticancer effects of methyl 2-(5-fluoro-2-hydroxyphenyl)-1H- benzo[d]imidazole-5-carboxylate (MBIC) with doxorubicin in breast cancer cells

Hasanpourghadi M, Abdul Majid N, Rais Mustafa M

PeerJ, 2018;6:e5577.
PMID: 30245930 DOI: 10.7717/peerj.5577

Combination Index (CI) analysis suggested that MBIC and doxorubicin synergistically inhibited up to 97% of cell proliferation in ER+/PR+MCF-7 and triple negative MDA-MB-231 breast cancer cell lines. Moreover, treatment of the breast cancer cells with the combined drugs resulted in lower IC50 values in contrast to the individual drug treatment. Small noncoding microRNAs (miRNA) may function as non-mutational gene regulators at post-transcriptional level of protein synthesis. In the present study, the effect of the combined treatment of MBIC and doxorubicin on the expression level of several miRNAs including miR-34a, miR-146a, miR-320a and miR-542 were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. These miRNAs have the potential to alter the protein level of survivin, the anti-apoptotic protein and reduce the metastatic activity in human breast cancer cell lines by interfering with the nuclear accumulation of NF-κB. Our results demonstrated the several fold changes in expression of miRNAs, which is drug and cell line dependent. This finding demonstrated a functional synergistic network between miR-34a, miR-320a and miR-542 that are negatively involved in post-transcriptional regulation of survivin in MCF-7 cells. While in MDA-MB-231 cells, changes in expression level of miR-146a was correlated with inhibition of the nuclear translocation of NF-κB. The overall result suggested that alteration in protein level and location of survivin and NF-κB by miR-34a, miR-320a, miR-146a and miR-542, remarkably influenced the synergistic enhancement of combined MBIC and doxorubicin in treatment of aggressive and less aggressive human breast cancer cell lines.
Fulltext The use of multiplex real-time PCR improves the detection of the bacterial etiology of community acquired pneumonia

Mustafa MI, Al-Marzooq F, How SH, Kuan YC, Ng TH

Trop Biomed, 2011 Dec;28(3):531-44.
PMID: 22433882 MyJurnal

Community-acquired pneumonia (CAP) is still a major cause of morbidity and mortality especially to children and compromised hosts, such as the old and those with underlying chronic diseases. Knowledge of pathogens causing CAP constitutes the basis for selection of antimicrobial treatment. Previous data have shown that etiological agents can be identified in only up to 50% of patients, but this figure can be improved by using polymerase chain reaction (PCR). This study was designed to evaluate multiplex real-time PCR as a method for rapid differential detection of five bacterial causes of CAP (Streptococcus pneumoniae, Burkholderia pseudomallei and atypical bacterial pathogens namely Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila) in CAP patients attending Hospital Tengku Ampuan Afzan (HTAA)/ Kuantan, Pahang, Malaysia. Two previously developed multiplex real-time PCR assays, duplex for the differential detection of S. pneumoniae and B. pseudomallei and triplex for the atypical bacterial pathogens, were used to detect a bacterial cause of CAP in blood and respiratory samples. Thus, 46 blood and 45 respiratory samples collected from 46 adult CAP patients admitted to HTAA were analysed by multiplex real-time PCR assays and conventional methods. The microbial etiology of CAP could be established for 39.1% (18/46) of CAP patients by conventional methods and this was increased to 65.2% (30/46) with the additional use of real-time PCR. The most frequently detected pathogens were S. pneumoniae (21.7% - all by PCR alone), Klebsiella pneumoniae (17.3%), B. pseudomallei (13% - 83% of them positive by PCR alone and 17% by both culture and PCR), Pseudomonas aeruginosa (6.5%), M. pneumoniae (6.5% - all by serology), C. pneumoniae (4.3% - all positive by both PCR and serology), L. pneumophila (2.1% - all by PCR alone), Escherichia coli (4.3%). Haemophilus infuenzae, Acinetobacter lwoffii and Acinetobacter baumannii were detected by conventional methods (2.1% for each).
Fulltext Transmitted drug resistance and antiretroviral treatment outcomes in non-subtype B HIV-1-infected patients in South East Asia

Phanuphak P, Sirivichayakul S, Jiamsakul A, Sungkanuparph S, Kumarasamy N, Lee MP, et al.

J Acquir Immune Defic Syndr, 2014 May 01;66(1):74-9.
PMID: 24413039 DOI: 10.1097/QAI.0000000000000108

BACKGROUND: We compared treatment outcomes of transmitted drug resistance (TDR) in patients on fully or partially sensitive drug regimens.
METHODS: Factors associated with survival and failure were analyzed using Cox proportional hazards and discrete time conditional logistic models.
RESULTS: TDR, found in 60 (4.1%) of 1471 Asian treatment-naive patients, was one of the significant predictors of failure. Patients with TDR to >1 drug in their regimen were >3 times as likely to fail compared to no TDR.
CONCLUSIONS: TDR was associated with failure in the context of non-fully sensitive regimens. Efforts are needed to incorporate resistance testing into national treatment programs.
Fulltext Trends in first-line antiretroviral therapy in Asia: results from the TREAT Asia HIV observational database

Boettiger DC, Kerr S, Ditangco R, Merati TP, Pham TT, Chaiwarith R, et al.

PLoS One, 2014;9(9):e106525.
PMID: 25184314 DOI: 10.1371/journal.pone.0106525

Antiretroviral therapy (ART) has evolved rapidly since its beginnings. This analysis describes trends in first-line ART use in Asia and their impact on treatment outcomes.
Fulltext Trends of CD4 cell count levels at the initiation of antiretroviral therapy over time and factors associated with late initiation of antiretroviral therapy among Asian HIV-positive patients

Kiertiburanakul S, Boettiger D, Lee MP, Omar SF, Tanuma J, Ng OT, et al.

J Int AIDS Soc, 2014;17(1):18804.
PMID: 24598459 DOI: 18804

INTRODUCTION: Although antiretroviral therapy (ART) has been rapidly scaled up in Asia, most HIV-positive patients in the region still present with late-stage HIV disease. We aimed to determine trends of pre-ART CD4 levels over time in Asian HIV-positive patients and to determine factors associated with late ART initiation.
METHODS: Data from two regional cohort observational databases were analyzed for trends in median CD4 cell counts at ART initiation and the proportion of late ART initiation (CD4 cell counts <200 cells/mm(3) or prior AIDS diagnosis). Predictors for late ART initiation and mortality were determined.
RESULTS: A total of 2737 HIV-positive ART-naïve patients from 22 sites in 13 Asian countries and territories were eligible. The overall median (IQR) CD4 cell count at ART initiation was 150 (46-241) cells/mm(3). Median CD4 cell counts at ART initiation increased over time, from a low point of 115 cells/mm(3) in 2008 to a peak of 302 cells/mm(3) after 2011 (p for trend 0.002). The proportion of patients with late ART initiation significantly decreased over time from 79.1% before 2007 to 36.3% after 2011 (p for trend <0.001). Factors associated with late ART initiation were year of ART initiation (e.g. 2010 vs. before 2007; OR 0.40, 95% CI 0.27-0.59; p<0.001), sex (male vs. female; OR 1.51, 95% CI 1.18-1.93; p=0.001) and HIV exposure risk (heterosexual vs. homosexual; OR 1.66, 95% CI 1.24-2.23; p=0.001 and intravenous drug use vs. homosexual; OR 3.03, 95% CI 1.77-5.21; p<0.001). Factors associated with mortality after ART initiation were late ART initiation (HR 2.13, 95% CI 1.19-3.79; p=0.010), sex (male vs. female; HR 2.12, 95% CI 1.31-3.43; p=0.002), age (≥51 vs. ≤30 years; HR 3.91, 95% CI 2.18-7.04; p<0.001) and hepatitis C serostatus (positive vs. negative; HR 2.48, 95% CI 1.-4.36; p=0.035).
CONCLUSIONS: Median CD4 cell count at ART initiation among Asian patients significantly increases over time but the proportion of patients with late ART initiation is still significant. ART initiation at higher CD4 cell counts remains a challenge. Strategic interventions to increase earlier diagnosis of HIV infection and prompt more rapid linkage to ART must be implemented.
Fulltext Tualang honey ameliorates viral load, CD4 counts and improves quality of life in asymptomatic human immunodeficiency virus infected patients

Wan Yusuf WN, Wan Mohammad WMZ, Gan SH, Mustafa M, Abd Aziz CB, Sulaiman SA

J Tradit Complement Med, 2019 Oct;9(4):249-256.
PMID: 31453119 DOI: 10.1016/j.jtcme.2018.05.003

This is the first study to report on the effects of honey in asymptomatic HIV positive subjects in ameliorating CD4 count, viral load (VL) and quality of life (QOL). It is a randomized, controlled, open labelled study, comparing the effects of Tualang honey (TH) administration for six months at three different doses: 20 g (THL), 40 g (THI) or 60 g (THH) daily compared with control (no administered treatment, THC). Only asymptomatic HIV positive subjects (n=95) having CD4 count 250-600 cell/ml, not on antiretrovirals were enrolled. Blood, (together with QOL questionnaires administration) were investigated at baseline, three and six months (CD4 cell count) while VL was determined only at baseline and six months. Significant reductions in CD4 counts in THL and THC groups (p= 0.003 for both) were seen with no significant reductions in the CD4 counts in THI and THH groups (p=0.447 and 0.053 respectively). There was improvement in VL in THC and THI (130% and 32% respectively) and reductions in THL and THH (26% and 8% respectively). Within and between group analyses for VL indicated significant differences between THL and THH compared to THC. In addition, significant improvement in QOL of groups which received TH was noted. TH has the potential to improve the QOL (physical and psychological) and CD4 counts. There was a trend of lower VL in asymptomatic HIV subjects following TH administration thus supporting the possible role of TH in boosting the immune system by improving CD4 counts, causing VL reductions in HIV positive subjects.
Fulltext Utilization of a Mixture Cure Rate Model based on the Generalized Modified Weibull Distribution for the Analysis of Leukemia Patients

Omer ME, Abu Bakar M, Adam M, Mustafa M

Asian Pac J Cancer Prev, 2021 Apr 01;22(4):1045-1053.
PMID: 33906295 DOI: 10.31557/APJCP.2021.22.4.1045

OBJECTIVE: Cure rate models are survival models, commonly applied to model survival data with a cured fraction. In the existence of a cure rate, if the distribution of survival times for susceptible patients is specified, researchers usually prefer cure models to parametric models. Different distributions can be assumed for the survival times, for instance, generalized modified Weibull (GMW), exponentiated Weibull (EW), and log-beta Weibull. The purpose of this study is to select the best distribution for uncured patients' survival times by comparing the mixture cure models based on the GMW distribution and its particular cases.
MATERIALS AND METHODS: A data set of 91 patients with high-risk acute lymphoblastic leukemia (ALL) followed for five years from 1982 to 1987 was chosen for fitting the mixture cure model. We used the maximum likelihood estimation technique via R software 3.6.2 to obtain the estimates for parameters of the proposed model in the existence of cure rate, censored data, and covariates. For the best model choice, the Akaike information criterion (AIC) was implemented.
RESULTS: After comparing different parametric models fitted to the data, including or excluding cure fraction, without covariates, the smallest AIC values were obtained by the EW and the GMW distributions, (953.31/969.35) and (955.84/975.99), respectively. Besides, assuming a mixture cure model based on GMW with covariates, an estimated ratio between cure fractions for allogeneic and autologous bone marrow transplant groups (and its 95% confidence intervals) were 1.42972 (95% CI: 1.18614 - 1.72955).
CONCLUSION: The results of this study reveal that the EW and the GMW distributions are the best choices for the survival times of Leukemia patients.
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Fulltext Virulence-associated genes and antibiotic resistance patterns of Vibrio spp. isolated from cultured marine fishes in Malaysia

Mohamad N, Amal MNA, Saad MZ, Yasin ISM, Zulkiply NA, Mustafa M, et al.

BMC Vet Res, 2019 May 28;15(1):176.
PMID: 31138199 DOI: 10.1186/s12917-019-1907-8

BACKGROUND: Vibriosis is an important bacterial disease of cultured marine fishes worldwide. However, information on the virulence and antibiotic resistance of Vibrio spp. isolated from fish are scarce. This study investigates the distribution of virulence associated genes and antibiotic resistance patterns of Vibrio spp. isolated from cage-cultured marine fishes in Malaysia.
RESULTS: A total of 63 Vibrio spp. isolated from 62 cultured marine fishes in various geographical regions in Peninsular Malaysia were analysed. Forty-two of the isolates (66.7%) were positive for all chiA, luxR and vhpA, the virulence genes produced by pathogenic V. harveyi. A total of 62 Vibrio isolates (98%) had tlh gene of V. parahaemolyticus, while flaC gene of V. anguillarum was detected in 43 of isolates (68%). Other virulence genes, including tdh, trh, hlyA and toxRvc were absent from any of the isolates. Multiple antibiotic resistance (MAR) was exhibited in all strains of Harveyi clade, particularly against ampicillin, penicillin, polypeptides, cephems and streptomycin. The MAR index ranged between 0.06 and 0.56, and 75% of the isolates have MAR index of higher than 0.20. Host species and geographical origin showed no correlation with the presence of virulence genes and the antibiotic resistance patterns of Vibrio spp.
CONCLUSIONS: The study indicates that majority of Vibrio spp. isolated from cultured marine fishes possess virulence genes, but were not associated with human pathogen. However, the antibiotics resistance is a real concern and warrants ongoing surveillance. These findings represent an updated knowledge on the risk of Vibrio spp. to human health, and also provides valuable insight on alternative approaches to combat vibriosis in cultured fish.
Fulltext Water Extract Have Superior Cytotoxic Effect Than Ethanolic Extract of Clinacanthus Nutans Leaves in Breast Cancer Stem Cells

Syarifah S, Rambe AS, Putra A, Ichwan M, Pane YS, Muhar AM, et al.

Acta Inform Med, 2023;32(1):4-10.
PMID: 38585603 DOI: 10.5455/aim.2024.32.4-10

BACKGROUND: The rapid development of medical technology in managing breast cancer patients still cannot solve the problem of recurrence and resistance. One of the causes of recurrence and molecular resistance is the presence of breast cancer stem cells (BCSCs). Clinacanthus nutans (C.nutans) is a plant found in Medan, North Sumatra, Indonesia. This plant is believed to have anticancer activity in community.
OBJECTIVE: Our study aimed to assess phytochemical of C.nutans leaves, isolate breast cancer stem cells and determine the cytotoxic effects of the ethanolic extract and water extract of C.nutans leaves on breast cancer stem cells at 24, 48, and 72 h of observation.
METHODS: We underwent the cytotoxic test by using MTT assay and isolated breast cancer stem cells by using MACS and validated them by mammosphere test.
RESULTS: We found alkaloids, flavonoids, glycosides and tannins in simplicia and all extracts. BCSCs was valid with the diameter of the mammosphere BCSCs was > 60 μm. The IC50 values of 100%, 60%, 40%, 20% EE, and WE of C.nutans leaves were 227.30; 46.05; 31.12; 98.54, and 16.16 μg/ml respectively in the first 24 hours. In administering WE of C.nutans leaves, BCSCs viability was decreased at 24,48 and 72 hours of observation, namely 69.29±26%; 75.82 ± 21.02% and 38.94±9.34 % (p < 0.0001).
CONCLUSION: The WE of C.nutans leaves had more substantial cytotoxic potential against BCSCs than the EE. The capability of WE C.nutans leaves to suppress BCSC's viability was time-dependent. The anticancer activity were believed originate from alkaloid and flavonoid group.

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