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  1. Fulltext Mesenchymal Stem Cell Expressing TRAIL as Targeted Therapy against Sensitised Tumour
    Fakiruddin KS, Ghazalli N, Lim MN, Zakaria Z, Abdullah S
    Int J Mol Sci, 2018 07 27;19(8).
    PMID: 30060445 DOI: 10.3390/ijms19082188
    Tapping into the ability of engineered mesenchymal stem cells (MSCs) to mobilise into the tumour has expanded the scope of cancer treatment. Engineered MSCs expressing tumour necrosis factor (TNF)-related apoptosis inducing ligand (MSC-TRAIL) could serve as a platform for an efficient and targeted form of therapy. However, the presence of cancer stem cells (CSCs) that are resistant to TRAIL and apoptosis may represent a challenge for effective treatment. Nonetheless, with the discovery of small molecular inhibitors that could target CSCs and tumour signalling pathways, a higher efficacy of MSC-TRAIL mediated tumour inhibition can be achieved. This might pave the way for a more effective form of combined therapy, which leads to a better treatment outcome. In this review, we first discuss the tumour-homing capacity of MSCs, its effect in tumour tropism, the different approach behind genetically-engineered MSCs, and the efficacy and safety of each agent delivered by these MSCs. Then, we focus on how sensitisation of CSCs and tumours using small molecular inhibitors can increase the effect of these cells to either TRAIL or MSC-TRAIL mediated inhibition. In the conclusion, we address a few questions and safety concerns regarding the utilization of engineered MSCs for future treatment in patients.
  2. Fulltext Chemo-Sensitization of CD133+ Cancer Stem Cell Enhances the Effect of Mesenchymal Stem Cell Expressing TRAIL in Non-Small Cell Lung Cancer Cell Lines
    Fakiruddin KS, Lim MN, Nordin N, Rosli R, Abdullah S
    Biology (Basel), 2021 Oct 26;10(11).
    PMID: 34827096 DOI: 10.3390/biology10111103
    Pre-clinical studies have demonstrated the efficacy of mesenchymal stem cells (MSCs) expressing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or MSC-TRAIL against several tumors. However, due to the existence of cancer stem cells (CSCs), some tumors, including non-small cell lung cancer (NSCLC), exhibit TRAIL resistance. This study was designed to evaluate the capacity of using first-line chemotherapies including cisplatin, 5-fluorouracil (5-FU) and vinorelbine to act as a chemo-sensitizer on CD133+ (prominin-1 positive) CSCs derived from NSCLC cell lines (A549, H460 and H2170) for the purpose of MSC-TRAIL-induced inhibition. We showed that MSC-TRAIL was resistant to all three chemotherapies compared to the NSCLC cell lines, suggesting that the chemotherapies had little effect on MSC-TRAIL viability. Pre-treatment using either cisplatin or 5-FU, but not with vinorelbine, was able to increase the efficacy of MSC-TRAIL to kill the TRAIL-resistant A549-derived CSCs. The study also demonstrated that both 5-FU and vinorelbine were an effective chemo-sensitizer, used to increase the anti-tumor effect of MSC-TRAIL against H460- and H2170-derived CSCs. Furthermore, pre-treatment using cisplatin was noted to enhance the effect of MSC-TRAIL in H460-derived CSCs; however, this effect was not detected in the H2170-derived CSCs. These findings suggest that a pre-treatment using certain chemotherapies in NSCLC could enhance the anti-tumor effect of MSC-TRAIL to target the CSCs, and therefore the combination of chemotherapies and MSC-TRAIL may serve as a novel approach for the treatment of NSCLC.
  3. Fulltext A comparative study of non-viral gene delivery techniques to human adipose-derived mesenchymal stem cell
    Abdul Halim NS, Fakiruddin KS, Ali SA, Yahaya BH
    Int J Mol Sci, 2014;15(9):15044-60.
    PMID: 25162825 DOI: 10.3390/ijms150915044
    Mesenchymal stem cells (MSCs) hold tremendous potential for therapeutic use in stem cell-based gene therapy. Ex vivo genetic modification of MSCs with beneficial genes of interest is a prerequisite for successful use of stem cell-based therapeutic applications. However, genetic manipulation of MSCs is challenging because they are resistant to commonly used methods to introduce exogenous DNA or RNA. Herein we compared the effectiveness of several techniques (classic calcium phosphate precipitation, cationic polymer, and standard electroporation) with that of microporation technology to introduce the plasmid encoding for angiopoietin-1 (ANGPT-1) and enhanced green fluorescent protein (eGFP) into human adipose-derived MSCs (hAD-MSCs). The microporation technique had a higher transfection efficiency, with up to 50% of the viable hAD-MSCs being transfected, compared to the other transfection techniques, for which less than 1% of cells were positive for eGFP expression following transfection. The capability of cells to proliferate and differentiate into three major lineages (chondrocytes, adipocytes, and osteocytes) was found to be independent of the technique used for transfection. These results show that the microporation technique is superior to the others in terms of its ability to transfect hAD-MSCs without affecting their proliferation and differentiation capabilities. Therefore, this study provides a foundation for the selection of techniques when using ex vivo gene manipulation for cell-based gene therapy with MSCs as the vehicle for gene delivery.
  4. Fulltext Human non-small cell lung cancer expresses putative cancer stem cell markers and exhibits the transcriptomic profile of multipotent cells
    Zakaria N, Yusoff NM, Zakaria Z, Lim MN, Baharuddin PJ, Fakiruddin KS, et al.
    BMC Cancer, 2015;15:84.
    PMID: 25881239 DOI: 10.1186/s12885-015-1086-3
    Despite significant advances in staging and therapies, lung cancer remains a major cause of cancer-related lethality due to its high incidence and recurrence. Clearly, a novel approach is required to develop new therapies to treat this devastating disease. Recent evidence indicates that tumours contain a small population of cells known as cancer stem cells (CSCs) that are responsible for tumour maintenance, spreading and resistant to chemotherapy. The genetic composition of CSCs so far is not fully understood, but manipulation of the specific genes that maintain their integrity would be beneficial for developing strategies to combat cancer. Therefore, the goal of this study isto identify the transcriptomic composition and biological functions of CSCs from non-small cell lung cancer (NSCLC).
  5. Fulltext Targeting of CD133+ Cancer Stem Cells by Mesenchymal Stem Cell Expressing TRAIL Reveals a Prospective Role of Apoptotic Gene Regulation in Non-Small Cell Lung Cancer
    Fakiruddin KS, Lim MN, Nordin N, Rosli R, Zakaria Z, Abdullah S
    Cancers (Basel), 2019 08 28;11(9).
    PMID: 31466290 DOI: 10.3390/cancers11091261
    Mesenchymal stem cells (MSCs) are emerging as vehicles for anti-tumor cytotherapy; however, investigation on its efficacy to target a specific cancer stem cell (CSC) population in non-small cell lung cancer (NSCLC) is lacking. Using assays to evaluate cell proliferation, apoptosis, and gene expression, we investigated the efficacy of MSCs expressing tumour necrosis factor (TNF)-related apoptosis inducing ligand (MSC-TRAIL) to target and destroy CD133+ (prominin-1 positive) NSCLC-derived CSCs. Characterization of TRAIL death receptor 5 (DR5) revealed that it was highly expressed in the CD133+ CSCs of both H460 and H2170 cell lines. The human MSC-TRAIL generated in the study maintained its multipotent characteristics, and caused significant tumor cell inhibition in NSCLC-derived CSCs in a co-culture. The MSC-TRAIL induced an increase in annexin V expression, an indicator of apoptosis in H460 and H2170 derived CD133+ CSCs. Through investigation of mitochondria membrane potential, we found that MSC-TRAIL was capable of inducing intrinsic apoptosis to the CSCs. Using pathway-specific gene expression profiling, we uncovered candidate genes such as NFKB1, BAG3, MCL1, GADD45A, and HRK in CD133+ CSCs, which, if targeted, might increase the sensitivity of NSCLC to MSC-TRAIL-mediated inhibition. As such, our findings add credibility to the utilization of MSC-TRAIL for the treatment of NSCLC through targeting of CD133+ CSCs.
  6. Fulltext The Effects of Lentivirus-Mediated Gene Silencing of RARβ on the Stemness Capability of Non-Small Cell Lung Cancer
    Abu Halim NH, Zakaria N, Theva Das K, Lin J, Lim MN, Fakiruddin KS, et al.
    J Cancer, 2021;12(12):3468-3485.
    PMID: 33995625 DOI: 10.7150/jca.50793
    Retinoic acid receptor beta is a nuclear receptor protein that binds to retinoic acid (RA) to mediate cellular signalling in embryogenic morphogenesis, cell growth, and differentiation. However, the function of RARβ in cancer stem cells (CSCs) has yet to be determined. This study aimed to understand the role of RARβ in regulating cell growth and differentiation of lung cancer stem cells. Based on the clonogenic assay, spheroid assay, mRNA levels of stem cell transcription factors, and cell cycle being arrested at the G0/G1 phase, the suppression of RARβ resulted in significant inhibition of A549 parental cell growth. This finding was contradictory to the results seen in CSCs, where RARβ inhibition enhanced the cell growth of putative and non-putative CSCs. These results suggest that RARβ suppression may act as an essential regulator in A549 parental cells, but not in the CSCs population. The findings in this study demonstrated that the loss of RARβ promotes tumorigenicity in CSCs. Microarray analysis revealed that various cancer pathways were significantly activated following the suppression of RARβ. The changes seen might compensate for the loss of RARβ function, CSCs population's aggressiveness, which led to the CSCs population's aggressiveness. Thus, understanding the role of RARβ in regulating the stemness of CSCs may lead to targeted therapy for lung CSCs.
  7. CRISPR/Cas9 in Stem Cell Research: Current Application and Future Perspective
    Patmanathan SN, Gnanasegaran N, Lim MN, Husaini R, Fakiruddin KS, Zakaria Z
    Curr Stem Cell Res Ther, 2018;13(8):632-644.
    PMID: 29895256 DOI: 10.2174/1574888X13666180613081443
    The clustered regularly interspaced short palindromic repeats-associated protein 9 or CRISPR/Cas9 system is one of the hottest topics discussed lately due to its robustness and effectiveness in genome editing. The technology has been widely used in life science research including microbial, plant, animal, and human cell studies. Combined with the pluripotency of stem cells, the technology represents a powerful tool to generate various cell types for disease modeling, drug screening, toxicology, and targeted therapies. Generally, the CRISPR/Cas9 system has been applied in genetic modification of pluripotent or multipotent stem cells, after which the cells are differentiated into specific cell types and used for functional analysis or even clinical transplantation. Recent advancement in CRISPR/Cas9 technology has widened the scope of stem cell research and its therapeutic application. This review provides an overview of the current application and the prospect of CRISPR/Cas9 technology, particularly in stem cell research and therapy.
  8. Fulltext Nucleofection optimization and in vitro anti-tumourigenic effect of TRAIL-expressing human adipose-derived mesenchymal stromal cells
    Fakiruddin KS, Baharuddin P, Lim MN, Fakharuzi NA, Yusof NA, Zakaria Z
    Cancer Cell Int, 2014;14(1):122.
    PMID: 25469108 DOI: 10.1186/s12935-014-0122-8
    Tumour homing capacity of engineered human adipose-derived mesenchymal stromal cells (ADMSCs) expressing anti-tumour agents might be the key for a much safer and yet efficient targeted tumour therapy. However, ADMSCs exhibit resistant to most gene transfection techniques and the use of highly efficient viral vectors has several disadvantages primarily concerning safety risk. Here, we optimized the use of highly efficient and safe nucleofection-based transfection using plasmid encoded for TNF-Related Apoptosis Inducing Ligand (TRAIL) into ADMSCs and investigated the potential anti-tumourigenic of TRAIL-expressing ADMSCs (ADMSCs-TRAIL) on selected cancer models in vitro.
  9. Fulltext Curcumin improves the efficacy of cisplatin by targeting cancer stem-like cells through p21 and cyclin D1-mediated tumour cell inhibition in non-small cell lung cancer cell lines
    Baharuddin P, Satar N, Fakiruddin KS, Zakaria N, Lim MN, Yusoff NM, et al.
    Oncol Rep, 2016 Jan;35(1):13-25.
    PMID: 26531053 DOI: 10.3892/or.2015.4371
    Natural compounds such as curcumin have the ability to enhance the therapeutic effectiveness of common chemotherapy agents through cancer stem-like cell (CSC) sensitisation. In the present study, we showed that curcumin enhanced the sensitivity of the double-positive (CD166+/EpCAM+) CSC subpopulation in non-small cell lung cancer (NSCLC) cell lines (A549 and H2170) to cisplatin-induced apoptosis and inhibition of metastasis. Our results revealed that initial exposure of NSCLC cell lines to curcumin (10-40 µM) markedly reduced the percentage of viability to an average of ~51 and ~54% compared to treatment with low dose cisplatin (3 µM) with only 94 and 86% in both the A549 and H2170 cells. Moreover, sensitisation of NSCLC cell lines to curcumin through combined treatment enhanced the single effect induced by low dose cisplatin on the apoptosis of the double-positive CSC subpopulation by 18 and 20% in the A549 and H2170 cells, respectively. Furthermore, we found that curcumin enhanced the inhibitory effects of cisplatin on the highly migratory CD166+/EpCAM+ subpopulation, marked by a reduction in cell migration to 9 and 21% in the A549 and H2170 cells, respectively, indicating that curcumin may increase the sensitivity of CSCs to cisplatin-induced migratory inhibition. We also observed that the mRNA expression of cyclin D1 was downregulated, while a substantial increased in p21 expression was noted, followed by Apaf1 and caspase-9 activation in the double-positive (CD166+/EpCAM+) CSC subpopulation of A549 cells, suggested that the combined treatments induced cell cycle arrest, therefore triggering CSC growth inhibition via the intrinsic apoptotic pathway. In conclusion, we provided novel evidence of the previously unknown therapeutic effects of curcumin, either alone or in combination with cisplatin on the inhibition of the CD166+/EpCAM+ subpopulation of NSCLC cell lines. This finding demonstrated the potential therapeutic approach of using curcumin that may enhance the effects of cisplatin by targeting the CSC subpopulation in NSCLC.
  10. 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.
  11. Fulltext Transplantation of human bone marrow mesenchymal stromal cells reduces liver fibrosis more effectively than Wharton's jelly mesenchymal stromal cells
    Rengasamy M, Singh G, Fakharuzi NA, Siddikuzzaman, Balasubramanian S, Swamynathan P, et al.
    Stem Cell Res Ther, 2017 06 13;8(1):143.
    PMID: 28610623 DOI: 10.1186/s13287-017-0595-1
    BACKGROUND: Mesenchymal stromal cells (MSCs) from various tissues have shown moderate therapeutic efficacy in reversing liver fibrosis in preclinical models. Here, we compared the relative therapeutic potential of pooled, adult human bone marrow (BM)- and neonatal Wharton's jelly (WJ)-derived MSCs to treat CCl4-induced liver fibrosis in rats.

    METHODS: Sprague-Dawley rats were injected with CCl4 for 8 weeks to induce irreversible liver fibrosis. Ex-vivo expanded, pooled human MSCs obtained from BM and WJ were intravenously administered into rats with liver fibrosis at a dose of 10 × 106 cells/animal. Sham control and vehicle-treated animals served as negative and disease controls, respectively. The animals were sacrificed at 30 and 70 days after cell transplantation and hepatic-hydroxyproline content, histopathological, and immunohistochemical analyses were performed.

    RESULTS: BM-MSCs treatment showed a marked reduction in liver fibrosis as determined by Masson's trichrome and Sirius red staining as compared to those treated with the vehicle. Furthermore, hepatic-hydroxyproline content and percentage collagen proportionate area were found to be significantly lower in the BM-MSCs-treated group. In contrast, WJ-MSCs treatment showed less reduction of fibrosis at both time points. Immunohistochemical analysis of BM-MSCs-treated liver samples showed a reduction in α-SMA+ myofibroblasts and increased number of EpCAM+ hepatic progenitor cells, along with Ki-67+ and human matrix metalloprotease-1+ (MMP-1+) cells as compared to WJ-MSCs-treated rat livers.

    CONCLUSIONS: Our findings suggest that BM-MSCs are more effective than WJ-MSCs in treating liver fibrosis in a CCl4-induced model in rats. The superior therapeutic activity of BM-MSCs may be attributed to their expression of certain MMPs and angiogenic factors.

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