ORIGINAL ARTICLE - Universiti Putra Malaysia...availability and low toxicity towards normal cell...

Mal J Med Health Sci 15(SUPP9): 66-73, Dec 2019 66

Malaysian Journal of Medicine and Health Sciences (eISSN 2636-9346)

ORIGINAL ARTICLE

Differential Antiproliferative Activity of Goniothalamin Against Selected Human Cancer Cell LinesSiti Aishah Abu Bakar1,2, Abdul Manaf Ali2, Nor Hazwani Ahmad1

1 Oncological and Radiological Sciences Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas, Penang, Malaysia.

2 Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Besut Campus, 22200 Besut, Terengganu, Malaysia

ABSTRACT

Introduction: Goniothalamin (GTN) is a plant styryl-lactone that has been reported to possess anticancer properties. However, limited studies were conducted to explore the selectivity of GTN to human cancer cells in comparison with normal human cells counterpart. The aim of this study was to evaluate the cytotoxicity and selectivity of GTN in human cancer cells lines of osteosarcoma cells (Saos-2), adenocarcinoma alveolar basal epithelial cells (A549), breast carcinoma cells (UACC-732), breast adenocarcinoma cells (MCF-7), colorectal adenocarcinoma cells (HT29). Human bone marrow-derived mesenchymal stem cells (HMSC) served as a control normal cell. Methods: The an-tiproliferative activity of GTN was assessed by MTT assay for 24, 48 and 72 h of incubation time. Then, the half maximal inhibitory concentration (IC50) and the selectivity index (S.I) were calculated to examine the potency and selectivity of GTN. Results: GTN has significantly exhibited concentration and time-dependent inhibitory effect to all tested cancer cells with the lowest IC50 values ranging from 0.62±0.06 to 2.01±0.28 µg/ml after 72 h. Besides, GTN was found to be better than doxorubicin, DOX (positive control) in term of its selectivity towards cancer cells as higher S.I values between 2.86±0.31 to 10.02±1.49 were determined for all the cancer cells. Conclusion: This study concludes that GTN fulfills the basic criteria as an effective therapeutic agent by possessing a very strong range of anticancer activity with less toxicity towards the normal cells. Thus, GTN could be considered as a promising alternative to present chemotherapeutic drug in cancer treatment.

Keywords: Antiproliferative; Cancer cells; Cytotoxicity; Goniothalamin; Selective index

Corresponding Author: Nor Hazwani Ahmad, PhDEmail: [email protected]: +6014-3076412/+604-5622530

INTRODUCTION

Cancer is considered the leading cause of death and the biggest obstacle to increasing life expectancy in the 21st century. There are approximately 18.1 million new cancer cases with 9.6 million cancer deaths estimated in 2018. The lung cancer is the most commonly diagnosed cancer in both sexes which is about 11.6% of the total cases followed by breast cancer in women, prostate cancer, and colorectal cancer, stomach cancer and liver cancer (1). The current cancer chemotherapy treatments are nonspecific which not only targeting the cancer cells but also the normal cells. Thus, there is an urgency to develop more effective and selective chemotherapeutic agents for cancer treatment with less toxicity to normal cells.

Nature especially plant is an inexhaustible source of novel bioactive compounds and its contribution

to the treatment and prevention of disease remains enormous, although recent synthetic chemistry has been overshadowed as a method of drug discovery (2). Plant-derived drugs are natural, readily available and less-toxic to normal human cells, so they are desired for anticancer treatment. At present, the examples of plant-derived purified compounds have been used clinically such as; paclitaxel from Taxus brevifolia Nutt. (Taxaceae), epipodophyllotoxin from Podophyllum spp. (Berberidaceae), vinblastine and vincristine from Catharanthus roseus (L.) (Apocynaceae), combretastatins from Combretum caffrum Kuntze (Combretaceae) and betulinic acid from Zizyphus spp. In addition, there are many other plant-derived products that have been studied for the further improvement and development of drugs (3). The growing interest in finding new plant-derived compounds offers opportunities for exploring its biological activity including anticancer properties. They are considered to be potential anticancer agents if they show the ability to kill multiples types of cancer, elucidating some mechanisms of action, good availability and low toxicity towards normal cell (4).

Goniothalamus is one of the largest genera of

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Mal J Med Health Sci 15(SUPP9): 66-73, Dec 2019

palaeotropical Annonaceae with over 160 species distributed in tropical Southeast Asia with the centre of diversity lies in Indochina and western Malaysia (5). There are many compounds have been isolated from Goniothalamus species especially styryl-pyrone and styryl-lactones such as altholactone, cardiopetalolactone, and goniothalamin. Goniothalamin (GTN) has been reported to possess a wide range of biological activities including anti-inflammatory, immunosuppressive and anticancer effects (6). The cytotoxic properties of GTN have been studied in a variety of cancer cell lines including leukaemia, breast, lung, oral, cervical, colon, ovarian, pancreatic and prostate cancer cells (7-22). GTN was also reported to have less toxicity to normal liver Chang cell (17), normal kidney cell (14) and normal mouse fibroblast (20). This compound has demonstrated in vitro potency but not yet tested and studies in a clinical setting. Although there are many studies have reported the anticancer properties of GTN; however, limited studies were conducted to explore the selectivity of GTN to human cancer cell lines in comparison to normal human cells. Besides, some other cancer cell lines including Saos-2 and UACC-732 have not been tested. In this study, the efficacy and selectivity of GTN were explored on several human cancer cell lines together on HMSC cells as a control. Two important parameters, the IC

50 and S.I were determined in order

to conclude the finding. The chemotherapeutic agent, doxorubicin (DOX) was used as a comparison to GTN activities. This study provides more evidence of the potential biological properties of GTN in consideration to be a promising alternative anticancer treatment in the future.

MATERIALS AND METHODS

Goniothalamin (GTN)Isolated GTN from the roots of Goniothalamus macrophyllus were kindly provided by Prof. Dr. Abdul Manaf Ali from Universiti Sultan Zainal Abidin (UNiSZA). The powdered-form compounds were dissolved in dimethylsulphoxide (DMSO) (Vivantis, USA) to get a stock solution of 10 mg/mL. The stock solution was then stored at -20 °C in aliquots for future use. The working solution of 60 µg/ml was prepared by diluting the stock solution with Phosphate Buffered Saline (PBS).

Cell LinesThere were five human cancer cell lines used in this study; osteosarcoma cells (Saos-2), adenocarcinoma alveolar basal epithelial cells (A549), breast carcinoma cells (UACC-732), breast adenocarcinoma cells (MCF-7), colorectal adenocarcinoma cells (HT29). Human bone marrow-derived mesenchymal stem cells (HMSC) were used as normal cells. All of the cell lines were originally obtained from the American Tissue Culture Collection (ATCC). They were grown in tissue culture flasks (Nunclon TM, Nunc) at 37°C, 5% CO

2 and 90%

humidity in complete DMEM medium (Sigma, USA),

containing 10% fetal bovine serum (Culture lab), penicillin (100 IU/ml) and streptomycin (100 µg/ml). The cells were grown confluence, which could be observed under an inverted microscope and subcultured when the cells reached 80% confluency.

Treatment of CellsA volume of 100 µl of 5 x 104 cells/ml was seeded into each well of 96-well plates overnight. The cells were then treated with different concentrations of GTN in double dilution manner ranging from 0 to 30 µg/ml. The treated cells were incubated for 24, 48 and 72 hours in a humidified 5% CO

2 incubator at 37oC.

MTT AssayThe cell viability was assessed by Cell Titer 96 Non-Radioactive Cell Proliferation Assay, MTT (Promega, USA) according to kit’s manual. Briefly, after the treatment of cell with GTN for 24, 48 and 72 hours, a volume of 20 µl MTT reagent was added to each well and incubated for 3 hours in a humidified 5% CO

2 incubator at 37oC. Then, the media was discarded

and 100 µl of stop solution was added to the wells and further incubated for 1 hour. The absorbance was measured at 570 nm with 630 nm as a reference using a microplate reader (BioTek, USA). The half maximal inhibitory concentration (IC

50) values were determined

from a graph of percentage cell viability versus the concentration of GTN. The IC

50 values were grouped

based on their range of activity; very strong : <5 µg/ml, strong : <5–10 µg/ml, moderate: 10–20 µg/ml, weak: 20–100 µg/ml and not active: >100 µg/ml [23,24]. SI values were calculated based on the ratio of IC

50 value

of normal cell line to IC50

value of cancer cell line. The SI value with more than 2 indicates that the particular compound produces selective toxicity towards cancer cells. On the other hand, the SI value with less than 2 indicates that the compound produces general toxicity with no selectivity between cancer and normal cells (25).

Morphological AssessmentThe morphological changes in cells with the strongest GTN activities; Saos-2 and MCF-7 cells were further examined. A volume of 100 µl of 5 x 104 cells/ml was seeded into each well of 96-well plates overnight and treated with GTN and DOX at IC

50. Time-lapse images

from 0 to 72 hours were automatically collected by IncuCyte ZOOM™ live cell imaging system (Essen BioScience, USA) at 10X magnification.

Statistical AnalysisAll experiments were performed in triplicate and each data represents mean ± standard deviation. For the cytotoxic activity of GTN in different cell lines at 24, 48 and 72 h with their corresponding group of toxicity from very potent cytotoxic to weak cytotoxic, the comparisons of the group were done using 2-way ANOVA, followed by the Bonferroni post-test for multiple comparisons.

68Mal J Med Health Sci 15(SUPP9): 66-73, Dec 2019

Whereas comparison between the treated cancer cells with the treated normal cells (HMSC) after 72h was done using one-way ANOVA, followed by the Dunnett’s post-test to detect any significant differences (* p<0.05; ** p<0.01; *** p< 0.001; ns not significant).

RESULTS

In general, GTN significantly exhibited concentration and time-dependent inhibitory effect to the treated cells as shown in Figure 1 (a - e). The percentage of cell viability decreased with the treatment of cells at the higher concentration of GTN and longer incubation time. The same pattern was found for the treatment of cells with the positive control, DOX (Figure 2, a-e).

Figure 1: Figure 1: Dose-response effects of GTN to different cell lines (a) Saos-2 (b) MCF-7 (c) UACC-732 (d) A549 (e) HT29 and (f) HMSC cells. The cytotoxicity of GTN was assessed by MTT assays at 24, 48 and 72 h. All experiments were performed in triplicate, and the data represent means ± standard deviations. Comparisons of the group were done by using 2-way ANOVA, followed by the Bonferroni post-test for multiple comparisons (* p<0.05; ** p<0.01; *** p<0.001).

The IC50

values were obtained from the cell viability graph following incubation time of 24, 48 and 72h and were summarized in Table I. For Saos-2 cells, incubation GTN for 24, 48 and 72 hours gave IC

50 values of 2.36±0.06,

1.18±0.15 and 1.29±0.08 µg/ml respectively. While the IC

50 values of DOX were 18.5±4.94, 0.32±0.03

and 0.27±0.01 µg/ml respectively. The IC50

values of GTN in MCF-7 cells were 4.65±0.75, 0.58±0.10 and 0.79±0.03 µg/ml for 24, 48 and 72 hours respectively. While the treatment of MCF-7 with DOX for 24, 48 and 72 h resulted in the IC

50 values of 1.1±0.29, 0.76±0.17

and 0.80±0.13 respectively. The treatment of GTN in UACC-732 cell for 24h gave a higher IC

50, more than

30 µg/ml compare to treatment with DOX, only 8.57 µg/

Figure 2: Dose-response effects of positive control, DOX to different cell lines (a) Saos-2 (b) MCF-7 (c) UACC-732 (d) A549 (e) HT29 and (f) HMSC cells. The cytotoxicity of GTN was assessed by MTT assays at 24, 48 and 72 h. All experiments were performed in triplicate, and the data represent means ± standard deviations. Comparisons of the groupwere done by using 2-way ANOVA, followed by the Bonferroni post-test for multiple comparisons (* p<0.05; ** p<0.01; *** p<0.001).

Table I: The IC50 values of GTN and DOX in different cell lines after 24, 48 and 72 h assessed by MTT assay

Cell Limes Incubation Time

Goniothalamin (0-30µg/ml) ± SD

Doxorubicin (0-30µg/ml) ± SD

SAOS-2 24H48H72H

2.36 ± 0.061.18 ± 0.151.29 ± 0.08

18.5 ± 4.940.32 ± 0.030.27 ± 0.01

MCF-7 24H48H72H

4.65 ± 0.750.58 ± 0.100.79 ± 0.03

1.1 ± 0.290.76 ± 0.170.80 ± 0.13

UACC-732 24H48H72H

>307.93 ± 0.422.01 ± 0.28

8.57 ± 1.830.57 ± 0.050.65 ± 0.09

A549 24H48H72H

15.0 ± 0.501.27 ± 0.150.62 ± 0.06

14.43 ± 3.040.27 ± 0.030.24 ± 0.01

HT29 24H48H72H

26.93 ± 2.1810.27 ± 1.541.64 ± 0.05

>300.34 ± 0.050.23 ± 0.01

HMSC 24H48H72H

11.6 ± 0.178.57 ± 0.816.23 ± 1.29

>307.0 ± 2.64

0.23 ± 0.04

All experiments were performed in triplicate, and the data represent means and standard deviations

ml. The incubation time of GTN for 48 and 72 h reduce the values of IC

50 to 7.93±0.42 and 2.01±0.28 µg/ml

respectively. The treatment of A549 cells with both GTN and DOX at 24h showed almost similar effects with the IC

50, 15 µg/ml. Whereas for an incubation time of 48

and 72 h, both of GTN and DOX resulted in the IC50

values below than 2 µg/ml. The IC

50 of GTN in HT29

cell for 24, 48 and 72 h were 26.93±2.18, 10.27±1.54

69



and 1.64±0.05 µg/ml respectively. The IC50

of GTN and DOX in normal HMSC cells after 72 h were 6.23 ±1.29 and 0.23±0.40 µg/ml respectively.

Figure 3 explained mainly two important aspects; 1) the significant comparison of the IC

50 values between

different incubation time, and 2) the classification of the IC

50 based on their range of activity from very potent

cytotoxic to weak cytotoxic. As mentioned earlier, different incubation time resulted in different IC

50 values

of GTN. Compare to incubation time at 48 and 72h, 24 h incubation of cancerous cells with GTN resulted in significantly higher IC

50 values (p< 0.001). For UACC-

732 and HT29 cells, incubation time at 48 h was also resulted in significantly higher IC

50 compared to 72 h.

However, treated Saos-2 cells, MCF-7 and A549 cells with GTN for 48 and 72 h give no significant different of IC

50 values. Based on the cytotoxicity classification,

GTN was considered a very potent cytotoxic agent in Saos-2 and MCF-7 cells (from 24 to 72 h) and other cancerous cell lines; UACC-732, A549 and HT29 cells (for 72h) with their IC

50 values were less than 5µg/ml.

However, the IC50

of GTN in normal HMSC cells was between strong to moderate (>5 to 20µg/ml).

The morphological of Saos-2 and MCF-7 cells were further examined since GTN was most effective in both cells for all incubation times. As shown in Figure 4 and 5, the untreated cells exhibited normal proliferation pattern and achieved confluency after 72 h. In contrast, the proliferation of treated cells were inhibited with considerable decrease in cell density for all incubation periods. Besides, there were prominent morphological changes including plasma membrane alterations in both treated cancer cell with GTN as well as DOX.

The selectivity index (SI) values which represent the ratio of the IC

50 value for normal cell line to the IC

50 value

for cancerous cell line after 72 hours of GTN and DOX treatments were summarized in Table II. The S.I value

Figure 3: The cytotoxic activity of GTN in different cell lines for 24, 48 and 72 H with their corresponding group of toxicity from very potent cytotoxic to weak cytotoxic. All experiments were done in triplicate, and the data represent means and standard deviations. Comparisons of the group were performed by using 2-way ANOVA, followed by the Bonferroni post-test for multiple comparisons (* p<0.05; ** p<0.01; *** p<0.001; ns not significant)

Figure 4: Morphological assessment of untreated and treated Saos-2 cells with GTN and DOX at IC50 for 24, 48 and 72h. The decrement in cell density and plasma membrane alterations (black arrows) were observed in GTN and DOX-treated cell. Phase images were captured using IncuCyte ZOOM™ live cell imaging system at 10X magnification

for Saos-2, MCF-7, UACC-9732, A549, HT29 cells were 5.04±0.64, 7.79±1.31, 2.86±0.31, 10.02±1.49 and 3.68±0.76 respectively. GTN selectively cytotoxic to all cancer cell lines with S.I values more than 2 for all the cancer cells. In contrast, DOX caused general toxicity with S.I value below than 2 to all type of cancer cell lines.

70Mal J Med Health Sci 15(SUPP9): 66-73, Dec 2019

using a light microscope and hemacytometer. Currently, metabolic activity-based tests such as MTT, MTS, WST, and LDH were preferred for the determination of cytotoxicity (26-28).

In the present study, MTT assay was used to explore the cytotoxic activity of GTN towards several cancer cell lines including Saos-2, MCF-7, UACC-732, A549 and HT29. The human mesenchymal stem cell was selected as the control normal cell in this study since their multilineage capabilities to differentiate into osteoblasts, adipocytes and chondrocytes (29,30). Besides, many studies have further reported their abilities to differentiate into multiple other cell types of mesodermal and non-mesodermal origin, including endothelial cells, cardiomyocytes, hepatocytes and neural cells (29). In addition, they are widely studied as cell-based drug delivery vectors in cancer therapy due to its homing capacity in almost all human cancer cell lines such as lung cancer,malignant glioma, breast cancer, colon carcinoma, pancreatic cancer, melanoma and ovarian cancer (31,32). Whereas for positive control, Doxorubicin was used because it is a broad antitumor spectrum and has been regarded as one of the most potent of chemotherapeutic drug (33). It is a nonselective class I anthracycline, and it has the ability to combat rapidly dividing cells by binding to DNA-associated enzymes, intercalate with DNA base pairs, and target multiple molecular targets to produce a range of cytotoxic effects on a variety of cancerous cells but also caused toxicity on noncancerous cells in the human body (33,34).

As shown in Figure 1, GTN showed a concentration and time-dependent inhibitory effects in the treated cells. The IC

50 values of GTN for each of the treated cells for 24, 48

and 72 h were summarized in Table 1. According to the United States National Cancer Institute Plant Screening Program, a pure compounds was considered to induce active cytotoxic effects if the IC

50 value in cancer cells,

following incubation between 48 and 72 hours is less than 4 µg/mL, while it is less than 20 µg/mL for crude extract (35,36). Besides, the range of activity of a certain compound is classified into several groups based on their IC

50 value as described followed; the IC

50 value of more

than 10-25 µg/mL is considered weak cytotoxicity, the IC

50 value of 5.0-10.0 µg/mL is classified as moderately

active and those compounds having less than 5.0 µg/mL is considered very active (23,24). As shown in Table 1, the cytotoxicity of GTN was almost as effective as a chemotherapic drug, DOX especially in Saos-2, MCF-7, and A549 cell lines. GTN was found very effective in killing Saos-2 and MCF-7 cells even at 24h incubation with GTN resulted in low of IC

50 value which is less

than 5 µg/ml. The proliferation of both cells were clearly inhibited by GTN as well as DOX with prominent plasma membrane alterations were observed after 24, 48 and 72h of incubation times (Figure 4 and 5). Other cancer cells like UACC-732, A549, and HT29 cells

Figure 5: Morphological assessment of untreated and treated MCF-7 cells with GTN and DOX at IC50 for 24, 48 and 72h. The decrement in cell density and plasma membrane alterations (black arrows) were observed in GTN and DOX-treated cell. Phase images were captured using IncuCyte ZOOM™ live cell imaging system at 10X magnification

Table II: The selectivity index (SI) which represents IC50 for normal cell line/IC50 for cancerous cell line after 72 hours of GTN and DOX treatment. High S.I value (>2) of a compound gives selective toxicity towards cancer cells. While the compound with S.I value <2 is con-sidered to give general toxicity in which it also can cause cytotoxicity in normal cells (25).

No. Cell Lines IC50

GTN IC50

DOX S.I GTN S.I DOX S.I GTN/S.I DOX

1 SAOS-2 1.29±0.08 0.27±0.01 5.04±0.64 0.84±0.12 5.96±0.18

2 MCF-7 0.79±0.03 0.80±0.13 7.79±1.31 0.29±0.02 27.05±4.58

3 UACC-732

2.01±0.28 0.65±0.09 2.86±0.31 0.35±0.01 8.13±0.93

4 A549 0.62±0.06 0.24±0.01 10.02±1.49 0.93±0.14 10.47±1.26

5 HT29 1.64±0.05 0.23±0.01 3.69±0.76 1.01±0.22 3.66±0.29

6 HMSC 6.23±1.29 0.23±0.04 - -

DISCUSSION

Cytotoxicity refers to the degree to which an agent has a specific destructive effect on certain cells. The evaluation of cytotoxicity is a critical point in a modern pharmaceutical development process and has been widely used by researchers for purposes, particularly in the screening of novel anticancer agents. It is an economical and safe way to obtain vital information on the biological properties of a new molecule that focuses on basic tolerability. In the past, the determination of cell cytotoxicity or viability was performed manually



were affected later, after 48h treatment. Interestingly, GTN was found very effective after 72 h in all treated cancer cells with IC

50 value between 0.62±0.06 to

2.01±0.28 µg/ml. For comparison, IC50

GTN in a normal cell, HMSC was 6.23±1.29µg/ml (moderate cytotoxic).

It is important to determine the selectivity index (S.I) of a compound in order to predict its selective action towards targeted cancer cells. The S.I value is a ratio of IC

50 value

for normal cell line to IC50

value for cancerous cell line. Higher S.I value indicates the more promising selectivity properties of a compound hold. However, an S.I below 2.0 indicates that while a compound may possess strong anticancer activity, it may be a general toxin (25,37). Interestingly, we found high S. I value for GTN for all the cancer cells (Saos-2, MCF-7, UACC-732, A549, and HT29) ranging from 2.86±0.31 to 10.02±1.49 compared to DOX, only between 0.29±0.02 to 1.01±0.22. The ratio of S.I GTN over S.I DOX, (S.I GTN/ S. I DOX) explained the fold change of GTN with respect to DOX. For MCF-7, GTN has possessed around 27 fold larger of selectivity as compared to DOX. Whereas for other tested cancer cells exhibited between 3 to 10 fold more effective than DOX. So we conclude that GTN is more selective towards cancer cells while DOX, in contrast, caused the general toxicity that might be very harmful to the normal cell as well.

This study corroborated with previous studies that reported cytotoxicity of GTN in numerous of cancer cell lines such as leukemic cells Jurkat T, K562, CEMSS and HL60 (7-10), liver cancer cell HepG2 and HCC (11,12), breast cancer cells MCF-7, T47D and MDA-MB-231 (13-15), lung cancer cells COR-L23, NCI-H460 and NSCLC (16-19), oral cancer cells Ca9-22 (20), cervical cancer HeLa (21), colon cancer cells HT29 and LS174T (14,17), ovarian cancer cell line Caov-3 (22), pancreatic cancer (PANC-1)(13) and prostate cancer cells PC-3 (16). Besides, GTN was also reported to have less toxicity to normal liver Chang cell (11), normal kidney cell (MDBK) (22) and normal mouse fibroblast (3T3) (14). Most of the treated cancer cells with GTN were reported to undergo various types of cell death, most notably apoptosis (17,20,38). The potential biomarker of GTN on several cancer cells has been mediated by its regulation of the cancer cell cycle and induction of apoptosis by caspase activation and oxidative stress (6). Efforts are needed to identify further pathways and therapeutic effects of this compound before entering the next stage of development.

CONCLUSION

Naturally-derived anticancer drugs are generally more preferable as they can overcome the non-specificity toxic effects on cells, which is the unavoidable limitation of chemotherapy. This study concludes that goniothalamin is an effective and selective therapeutic agent with a very strong range of cytotoxicity against cancer cells (Saos-2,

MCF-7, UACC, A549, HT29) and less toxicity towards the normal cells (HMSC). The selective inhibitory effects indicate that goniothalamin carries unique molecular properties that has the ability to induce cytotoxicity effects on specific cells. The overall findings have driven to the importance of further investigating the potential of goniothalamin for the development of novel cancer-specific anticancer agent. ACKNOWLEDGEMENTS

This work was partly supported by Bridging Grant 2018 (Project code: 6316369) and Short Term Research Grant 2019 (Project code: 6315268) from Universiti Sains Malaysia.. SAAB is supported by the Skim Latihan Akademik Bumiputera (SLAB) scholarship, under the Ministry of Education Malaysia-Higher Education.

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