Journal of Medical Research and Development (JMRD)

Jul. 2013, Vol. 2 Iss. 3, PP. 48-54

Insulin Sensitivity Enhancement of the Mixture of Tinospora Crispa and Gelam (Melaleuca Cajuputi) Honey and Its Antiproliferative Activity on Hepatocellular carcinoma, HepG2: A Preliminary Study
Mohd Nazri Abu1, Muhammad Ashraf Mohd Salleh2, Nabilatul Hani Mohd Radzman2,
Wan Iryani Wan Ismail*2, Rosmadi Mohd Yusoff2, Hamzah Fansuri Hasan1 1 Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia 2 Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia * w_iryani@salam.uitm.edu.my
Abstract- This study aims to investigate the effect of Tinospora crispa and Gelam (Melaleuca cajupati) honey mixture on human hepatocellular carcinoma (HepG2) and normal human hepatocytes (WRL-68) cell lines. Cell cytotoxicity was measured using MTS assay. The IC50 obtained was 42.67% in HepG2 and none in the WRL-68. Western blotting showed an increment in phosphorylation of tyrosine (PY20), indicating induction of Caspase 3 apoptotic proteins, whereas, the gene product of insulin growth-like receptor (IGF-1R) was reduced, indicating cancer cell inhibition. It is concluded that the elevation of Caspase 3 and the inhibition of IGF-1R indicates the ability of this mixture to simultaneously promote apoptosis and sensitized insulin in HepG2 cells. Keywords- Tinospora crispa; Gelam Honey; Apoptosis; Cytotoxicity; Insulin Sensitivity; Hepg2; WRL-68

I. INTRODUCTION Obesity, diabetes mellitus, and cancer are the health conditions that becoming more prevalent globally. These conditions have been linked to insulin-resistance and therefore involve the insulin signalling pathway [1-3]. A study revealed that 35% people taking inappropriate daily diet is among the factor that contributes to the diseases [4]. Therefore, many are now turning to alternative therapies including taking natural product as a part of their diet since there are still no specific cures for each of these conditions. For instance, mixture of Tinospora crispa and Gelam (Melaleuca cajupati) honey has been consumed in traditional Malaysian community as a form of health drink. However, the cytotoxic effect of the mixture has not been determined. Thus, in this study, the cytotoxic effects of the mixture on human hepatocellular carcinoma (HepG2) and normal liver cell lines (WRL-68), as well as its effect on Caspase 3 and IGF-1R expression were investigated. This investigation protocol allowed evaluation of the effectiveness of the mixture in preventing cancer cells growth as well as its effect on normal cells line. II. LITERATURE REVIEW A. Insulin Signalling Pathway Insulin is an important hormone that is synthesized and secreted by the pancreatic -cells of the islets of Langerhans. It plays major metabolic roles in the body as it maintains glucose homeostasis by controlling glucose uptake and utilization in the insulin signalling pathway. Hence, dysfunction in the system or pathway may lead to metabolic disorders such as diabetes mellitus and cancer [5, 6]. The most common impairment of the insulin signalling pathway is insulin resistance. A dysfunctional insulin signalling pathway will lead to hyperglycaemia, where glucose levels in the blood is higher than normal as glucose cannot be taken up into the cells. In order to compensate, insulin will be continuously secreted, thus contributing to elevated insulin levels or hyperinsulinemia [7, 8]. Although the actual factors that lead to insulin resistance in cancer patients are yet to be established, it was demonstrated that insulin resistance is directly correlated to cancer or tumour progression in hyperinsulinaemic models [9, 10]. B. Tinospora crispa Tinospora crispa (L.) Hook. F. & Thomson, from the Menispermaceae family is a climbing plant that can be found in many

- 48 -

Journal of Medical Research and Development (JMRD)

Jul. 2013, Vol. 2 Iss. 3, PP. 48-54

Asian tropical and subtropical regions including Malaysia, Thailand, Indonesia and India. This plant has various synonyms such as Brotowali, Akar Seruntun, Andawali and Patawali [11] [12]. This plant has been used widely by traditional health practitioners to treat, among others, diabetes, fever and hypertension [11]. The antidiabetic effect of T. crispa was reported in the observation of increased intestinal glucose uptake and the evocation of insulin release upon treatment with the plants extracts [13]. Furthermore, the effect of T. crispa on the Imprinting Control Region (ICR) diabetic mice revealed activation of insulin receptor-AKT-GLUT2 expression and insulin sensitivity enhancement by borapetoside c which contributed to the hypoglycaemic effect in-vivo [14]. Other reported medicinal properties of T. crispa include antioxidant and antiproliferative activities against human breast cancer cell line, MCF7, and antimalarial activity [13, 15]. Tumour and cancer progression can be caused by several factors such as excessive exposures to chemicals and radiation, and also from the imbalance of oxidants - antioxidants levels [16]. Therefore, introducing antioxidants to neutralize this imbalance may inhibit their progression, and would be preferable compared to radiotherapy and surgery. The utilization of T. crispa for this purpose is logical as the plants methanolic extracts contain high level of flavonoids and phenolic compounds [17, 18]. C. Gelam (Melaleuca cajupati) Honey Honey is a natural compound derived from floral sources containing carbohydrates such as fructose, glucose and sucrose, together with vitamins and minerals [19]. Honey has gained popularity worldwide especially among diabetics to be used as an alternative to normal sucrose due to its low glycaemic index. These constituents of honey helps inhibit bacterial growth and contributes towards its stability and convenience of storage. In particular, the Malaysian honey from the Gelam (M. cajupati) tree, or in short, Gelam honey, have been observed to inhibit growths of Escherichia coli and Staphylococcus aureus, as well as containing non-peroxide factors like phenolic acids [20, 21]. Gelam honey also exhibits high potentials as an antioxidant due to the levels of flavonoids and phenolic compounds which contribute to its free radical scavenging activities [20]. Furthermore, the antiproliferative effects of Gelam honey through induction of DNA damage and apoptosis of colon cancer cells have also been reported [18, 22]. Extracts of Gelam honey have also been tested in-vitro against inflammatory agents and cytokines such as tumor necrosis factor- (TNF-) where it reduces their actions, posing as an anti-inflammatory agent [23]. The fact that Gelam honey can exert its effects on the activities of TNF- also means that it can potentially sensitize insulin actions as TNF- is one of the contributing factors of insulin resistance [24]. Both natural products that were utilized in this study have been used since ancient time among Malay community as a drink for maintaining health. The fact that both were reported to exhibit antioxidant and antiproliferative properties, as well as potentially improve insulin resistance and induce apoptosis was the main impetus for conducting this study. III. EXPERIMENTAL A. Cell Culture Human hepatocellular carcinoma (HepG2) and normal liver cells (WRL-68) were used in the study. Cells were maintained in Minimum Essential Medium (MEM) supplemented with 10% foetal bovine serum (FBS) and 1% of each penicillinstreptomycin solution and gentamicin. Cells were incubated and maintained in 5% carbon dioxide saturation at 37C. B. Plant Material and Extract Procedure The T. crispa plants were collected in Muar, Malaysia and identified by Forest Research Institute of Malaysia (FRIM) on sample labelled SBD013/11. The plants were than processed by Ta Mim Jaya Enterprise (Ta Mim Herbs). The stems were cut into smaller sections and the unwanted parts removed, left to dry and were grounded into powder form. The crude plant powder and Gelam (M. cajupati) honey was then combined and soaked in methanol with constant stirring at room temperature until exhausted. This mixture was then filtered and the filtrates were evaporated using a rotary evaporator. Dried extracts were kept at -80C until further analysis. Prior to tests, the extract was diluted in Minimum Essential Medium (MEM) to form stock solution with a concentration of 1 mg/mL. Working solutions were formulated in different dilutions with MEM to create a series of 100%, 50%, 25% and 12% concentrations. C. Total Phenolic Content (TPC) Assay Folin-Ciocalteu method described by Azlim Almey et. al.,[25] was adapted to analyze the total phenolic content of the mixture extract. Different concentrations of Gallic acid were used as standards. The Folin-Ciocalteu reagent was diluted tentimes to prepare the working solution. Standards and samples were added to 255 L of the Folin-Ciocalteu working solution

- 49 -

Journal of Medical Research and Development (JMRD)

Jul. 2013, Vol. 2 Iss. 3, PP. 48-54

and allowed to stand in the dark at room temperature. Next, 255 L of 6% (w/v) sodium carbonate (Na2CO3) were added to each tube and allowed to stand for 90 minutes. Absorbance values were measured at 725 nm using Ultrospec 2100pro (Biochrom, Cambridge) UV-Visible spectrophotometer and TPC values were determined from the standard calibration curve of Gallic acid. D. Cytotoxity Assay In order to study cytotoxicity activity of the extract, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS) assay was conducted [18, 26]. Cells were seeded onto 96-well plates at a concentration of 2104 cells/ml and were left to attach for 24 hours. Cells were then treated with 100%, 50%, 25% and 12% concentrations of the extract for 72 hours. Next, 20 L of MTS solution was added to each well and incubated under normal culture conditions for two hours. Absorbance values corresponding to the formazan products were then recorded at the wavelength of 490nm using Sunrise (Tecan, Austria) plate reader. E. Western Blotting Samples (2104 cells/ml) harvested after treatments were added with Laemmlis loading buffer, boiled for 5 minutes and subjected to 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at 308 V for one hour. Electroblotting to nitrocellulose membrane followed for another hour at 100 V [27]. Standard immunoblotting procedures were performed using phosphotyrosine (PY20), insulin-like growth factor 1 receptor (IGF-1R) and Caspase-3 monoclonal antibodies similar to those described by Chau et.al [28] with minor adjustments. Data were later analyzed using Image J (Image processing and analysis in Java software) [29]. IV. RESULTS A. Total Phenolic Content (TPC) Total phenolic content (TPC) value of the methanolic extracts of T. crispa and Gelam honey was expressed in milligram Gallic acid per 100 mg of sample. The total phenolic content obtained was 0.9832 mg GAE (Gallic acid equivalent)/100 mg of the methanolic extract of T.crispa and Gelam honey. B. MTS Assay Figure 1 showed the effect of the methanolic extract of T. crispa and Gelam honey mixture on the proliferation of HepG2 and WRL-68 cells. It was shown that the percentage of cell death increases with the increasing concentration of extract concentration. The IC50 of the extract on HepG2 cells was 42.67% while in the normal liver cells, WRL-68, no IC50 was observed. Even at the highest concentration of extract (100%), the percentage of cell death in the WRL-68 did not reach 50% of total cells.

Fig. 1 Effect of the methanolic extract of T. crispa and Gelam honey mixture on the proliferation of HepG2 (a) and WRL-68 (b) cells

C. Western Blot Phosphotyrosine (PY20) protein was over-expressed in HepG2 cells treated with 50% of extract concentration. However, this expression did not exceed expression level in the positive control (Figure 2). It was also shown that the expression of insulin-like growth factor receptor 1 (IGF-1R) decreases with the increase in extract concentration.

- 50 -

Journal of Medical Research and Development (JMRD)

Jul. 2013, Vol. 2 Iss. 3, PP. 48-54

Fig. 2 Expressions of proteins as observed from Western Blotting in HepG2 cancer cell lines

Bands formation corresponds to protein intensities. 0.5 mM Doxorubicin was used as the positive control. PY20 and IGF1R were targeted in relation to the insulin signalling pathway. Caspase 3 and also IGF-1R represent apoptosis activities and actin acts as a housekeeping protein. V. DISCUSSIONS As previously stated, the Folin-Ciocalteu method was used in order to measure total phenolic content of the extract in question. The total phenolic content recorded was 0.9832 mg GAE (Gallic acid equivalent)/100 mg which are considered high. Phenolic compounds are highly associated with antioxidant activities especially those that are acquired naturally from plants and foods. In addition, they are also said to be able to block free radical damage thus may act as preventive agents in the development of several diseases like cancer [30]. Based on the high total phenolic content of the methanolic extract of T. crispa and Gelam honey mixture, it is suggested that this extract may have potent antioxidant activity. However, further antioxidant assays are needed to confirm this hypothesis. Although the Folin-Ciocalteu method has been regularly employed in the investigation of phenolic and polyphenol antioxidants, it was also found not to be highly specific in the detection of phenolic compounds, as it also accounted for several other reducing substances [31]. MTS assays have been conducted to evaluate the cytotoxicity activity of the extract apart from studying their antiproliferative effects towards the HepG2 cancer cells. The principle behind this experiment is that the MTS tetrazolium salt will be converted to soluble formazan products via the action of mitochondrial dehydrogenase enzyme in viable cells. This means that the level of formazan production is significantly proportional to the number of cells that survived the treatments, and also proportional to the absorbance value [32]. As indicated previously, both cancer and normal cell lines were given the same series of treatment concentrations for a specific 72 hours incubation time. Referring to Figure 1(a), HepG2 hepatocellular carcinoma cell lines shows elevated cell death rate upon the increase in treatment concentrations. An IC50 value of 42.67% can be deduced from the plotted cell death curve, in Figure 1(a). This shows that the half maximal inhibitory concentration of the mixture extracts towards the HepG2 cell line is 42.67%, where 50% of the total cells present are killed at this point. This suggests that HepG2 might undergo apoptosis, induced by the treatments with T. crispa and Gelam honey mixture extracts Figure 1(b) shows the percentages of cell deaths in normal liver cell line, WRL-68 which remained low, even after treatments with 100% concentration of the extract of interest. Since liver plays vital roles in metabolism, protein synthesis and detoxification, it is important to investigate the effects of the mixture extracts on normal liver cell line. Since the percentage of cell death did not reach 50% throughout the increasing series of treatment concentrations and no IC50 was observed, therefore, it is assumed that the extract is not toxic to normal liver cells. However, more research is needed confirm this. Western blotting showed an increased in PY20 level upon treatments implying that insulin sensitivity could be enhanced. PY20 is a phosphotyrosine which elevates upon phosphorylation of the insulin receptor and its substrate during the insulin signalling pathway for the uptake of extracellular glucose [27, 33]. Therefore, the increase in PY20 expression is parallel with the increase in insulin binding and insulin sensitivity in the cells. The decline in IGF-1R expressions with the increase of treatment concentration based on the Western Blot analysis is worth noting. IGF-1R is a potent activator of the phosphatidyl inositol 3 kinase (PI3K)-Akt signalling pathway and it is also an inhibitor of apoptosis or programmed cell death [34]. Hence, the increase of IGF-1R may also contribute to chemotherapy resistance. The inclinations in IGF-1R levels is important in combating cancer, and this correlates closely with the results obtained where its levels were significantly reduced in HepG2 treated at a concentration of 50% of the extract. Nevertheless,

- 51 -

Journal of Medical Research and Development (JMRD)

Jul. 2013, Vol. 2 Iss. 3, PP. 48-54

this did not exceed the effects of doxorubicin as the positive control, as the IGF-1R levels observed were untraceable, thus, the extracts may not be as potent as doxorubicin in preventing the inhibition of apoptosis via reducing IGF-1R levels. Apart from IGF-1R, another protein that has a crucial role in the apoptotic pathway is Caspase 3. Caspase 3 is responsible for both intrinsically and extrinsically activated apoptosis via actions of Caspase 9 in the former and Caspase 8 in the latter [35]. Apart from that, caspases were also suggested to have a central role in the morphological features of apoptosis by activating each other and will eventually lead to precise caspase cascades [36]. The results presented in Figure 2 support this fact as Caspase 3 levels were seen to have been elevated with the increase in treatment concentrations, which also went hand in hand with those treated with doxorubicin. It is assumed that apoptosis was induced in the HepG2 hepatocellular carcinoma cells, potentially causing and complementing in elevated cell deaths (Figure 1). This also suggests that the combination of T. crispa and Gelam honey extracts can potentially kill cancer cells via the activation of the apoptotic pathway. VI. CONCLUSION In summary, combination of T. crispa and Gelam honey mixture possesses potent antioxidant activity as well as moderate antiproliferative activity against human hepatocellular carcinoma cells, HepG2, depending on treatment concentration. The methanolic extract of the combined natural products possess high total phenolic content which contributed to its potent antioxidant and antiproliferative activities. This fact was also supported by the elevation of Caspase 3 and the reduction in IGF-IR protein expression. Furthermore, the extracts may improve insulin sensitizing activity, based on its potential to increase the expression of PY20. However, these findings need to be authenticated further, particularly using in vivo system, in order to prove its effectiveness as a dietary adjunct in healthcare maintenance.
ACKNOWLEDGEMENTS

The current research was funded by the Fundamental Research Grant Scheme (FRGS) under Ministry of Higher Education Malaysia, 2011 for the project: 600-RMI/FRGS 5/3 (43/2011) and Dana Kecemerlangan under Ministry of Higher Education Malaysia, 2011 for the project: 600-RMI/ST/DANA 5/3/Dst (394/2011).
REFERENCES [1] Mohammaed Qatanani and L. Mitchell A, "Mechanism of obesity-associated insulin resistance: many choices on the menu," Genes & Development, pp. 1443-1445, 2007.

[2] E. J. Gallagher, Y. Fierz, R. D. Ferguson, and D. LeRoith, "The pathway from diabetes and obesity to cancer, on the route to targeted therapy," Endocr Pract, vol. 16, pp. 864-73, 2010.
[3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] P. K. Shah, "Innate immune pathway links obesity to insulin resistance," American Heart Association, 2007. Y. J. Surh, "Cancer chemoprevention with dietary phytochemicals," Nat Rev Cancer, vol. 3, pp. 768-80, 2003. E. Gonzalez, E. Flier, D. Molle, D. Accili, and T. E. McGraw, "Hyperinsulinemia leads to uncoupled insulin regulation of the GLUT4 glucose transporter and the FoxO1 transcription factor," Proc Natl Acad Sci U S A, vol. 108, pp. 10162-7, 2011. J.-R. Zhou, G. L. Blackburn, and W. A. Walker, "Symposium introduction: metabolic syndrome and the onset of cancer," The American Journal of Clinical Nutrition, vol. 86, pp. 817S-819S, September 1, 2007 2007. I. F. Godsland., "Insulin resistance and hyperinsulinemia in the development and progression of cancer," Clinical science, pp. 315-332, 2010. M. H. Shanik, Y. Xu, J. krha, R. Dankner, Y. Zick, and J. Roth, "Insulin Resistance and Hyperinsulinemia: Is hyperinsulinemia the cart or the horse?," Diabetes Care, vol. 31, pp. S262-S268, 2008. B. Balkau, H. S. Kahn, D. Courbon, E. Eschwege, and P. Ducimetiere, "Hyperinsulinemia predicts fatal liver cancer but is inversely associated with fatal cancer at some other sites: the Paris Prospective Study," Diabetes Care, vol. 24, pp. 843-9, 2001. T. Yoshikawa, Y. Noguchi, C. Doi, T. Makino, and K. Nomura, "Insulin resistance in patients with cancer: relationships with tumor site, tumor stage, body-weight loss, acute-phase response, and energy expenditure," Nutrition, vol. 17, pp. 590-593, 2001. A. C. Dweck and J.-P. Cavin, "Andawali (Tinospora crispa) a review," Personal care megazine, vol. 7, pp. 1-3, 2006. S. Mohamad, N. M. Zin, H. A. Wahab, P. Ibrahim, S. F. Sulaiman, A. S. Zahariluddin, and S. S. Noor, "Antituberculosis potential of some ethnobotanically selected Malaysian plants," J Ethnopharmacol, vol. 133, pp. 1021-6, 2011. H. Noor and S. J. H. Ashcroft, "Pharmacological characterisation of the antihyperglycaemic properties of Tinospora crispa extract," Journal of Ethnopharmacology, vol. 62, pp. 7-13, 1998. C. T. Ruan, S. H. Lam, T. C. Chi, S. S. Lee, and M. J. Su, "Borapetoside C from Tinospora crispa improves insulin sensitivity in diabetic mice," Phytomedicine, vol. 19, pp. 719-24, 2012. A. R. N. Najib Nik, T. Furuta, S. Kojima, K. Takane, and M. Ali Mohd, "Antimalarial activity of extracts of Malaysian medicinal plants," J Ethnopharmacol, vol. 64, pp. 249-54, 1999. H. Kikuzaki, M. Hisamoto, K. Hirose, K. Akiyama, and H. Taniguchi, "Antioxidant properties of ferulic acid and its related compounds," J Agric Food Chem, vol. 50, pp. 2161-8, 2002. M. Ibrahim, W. Wan-Nor Izzah, A. Narimah, Z. Nurul Asyikin, and S. Siti-Nur Shafinas, "Anti-proliperative and antioxidant effects of Tinospora crispa (Batawali)," Biomedical Research vol. 22, pp. 57-62, 2011. A. Mohd Nazri, M. S. Muhammad Ashraf, E. Zolkapli, H. H. Mizaton, H. Hamzah Fansuri, and W. I. Wan Iryani, "Anti-proliferative effect of Tinaspora crispa (L.) Hook. F. & Thompson and Gelam (Melaleuca sp.) honey on several cancer cell lines," in Business, Engineering and Industrial Applications (ISBEIA), 2011 IEEE Symposium on, 2011, pp. 545-548. S. Bogdanov, "Honey composition," Bee product science, pp. 1-13, 2009. A. M. Aljadi and M. Y. Kamaruddin, "Evaluation of the phenolic contents and antioxidant capacities of two Malaysian floral honeys," Food Chemistry,

[19] [20]

- 52 -

Journal of Medical Research and Development (JMRD)

Jul. 2013, Vol. 2 Iss. 3, PP. 48-54

vol. 85, pp. 513-518, 2004. [21] A. M. Aljadi and M. Y. Kamaruddin, "Isolation and Identification of Phenolic Acids in Malaysian Honey with Antibacterial Properties," Turk J Med Sci, vol. 33 pp. 229-236, 2002. [22] C. T. Wen, S. Z. Hussein, S. Abdullah, N. A. Karim, S. Makpol, and Y. A. Mohd Yusof, "Gelam and Nenas honeys inhibit proliferation of HT 29 colon cancer cells by inducing DNA damage and apoptosis while suppressing inflammation," Asian Pac J Cancer Prev, vol. 13, pp. 1605-10, 2012. [23] M. Kassim, M. Achoui, M. R. Mustafa, M. A. Mohd, and K. M. Yusoff, "Ellagic acid, phenolic acids, and flavonoids in Malaysian honey extracts demonstrate in vitro anti-inflammatory activity," Nutr Res, vol. 30, pp. 650-9, 2010. [24] G. S. Hotamisligil, "Inflammatory pathways and insulin action," Int J Obes Relat Metab Disord, vol. 27, pp. S53-5, 2003. [25] A. A. Azlim Almey, C. Ahmed Jalal Khan, I. Syed Zahir, K. Mustapha Suleiman, M. R. Aisyah, and K. Kamarul Rahim, "Total phenolic content and primary antioxidant activity of methanoloic and ethanolic extracts of aromatic plants' leaves," International Food Research Journal, vol. 17, pp. 10771084, 2010. [26] N. D. Henry and P. A. Fair, "Comparison of in vitro cytotoxicity, estrogenicity and anti-estrogenicity of triclosan, perfluorooctane sulfonate and perfluorooctanoic acid," J Appl Toxicol, vol. 33, pp. 265-72, 2013. [27] W. I. Ismail and T. S. Pillay, "Insulin resistance induced by antiretroviral drugs: Current understanding of molecular mechanisms," JEMDSA vol. 14, pp. 129-132, 2009. [28] H. Chau, C. Mirtsos, and H. L. Huang, "Regulation of death complexes formation in tumor necrosis factor receptor signaling," Exp Cell Res, vol. 317, pp. 1841-50, 2011. [29] National Institutes of Health US. (2004). ImageJ (Image Processing and Analysis in Java). Available: http://rsb.info.nih.gov/ij/ [30] A. E.-M. M. R. Afify, H. S. El-Beltagi, S. M. A. El-Salam, and A. A. Omran, "Biochemical changes in phenols, flavonoids, tannins, vitamin E, carotene and antioxidant activity during soaking of three white sorghum varieties," Asian Pacific Journal of Tropical Biomedicine, vol. 2, pp. 203-209, 2012. [31] V. L. Singleton, R. Orthofer, and R. M. Lamuela-Ravents, "[14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent," in Methods in Enzymology. vol. Volume 299, P. Lester, Ed., ed: Academic Press, 1999, pp. 152-178. [32] Promega, "Cell Titer 96 AQueous non-radioactive cell proliferation assay (MTS)," ed: Promega Pt Ltd, 2013. [33] W. I. Ismail, J. A. King, K. Anwar, and T. S. Pillay, "Indinavir and nelfinavir inhibit proximal insulin receptor signalling and salicylate abrogates inhibition: Potential role of the NFkappa B pathway," J Cell Biochem, vol. 5, p. 24513, 2013. [34] J. Yeh, J. Litz, P. Hauck, D. L. Ludwig, and G. W. Krystal, "Selective inhibition of SCLC growth by the A12 anti-IGF-1R monoclonal antibody correlates with inhibition of Akt," Lung Cancer, vol. 60, pp. 166-74, 2008. [35] M. Nagase, T. Shiota, A. Tsushima, M. Murshedul Alam, S. Fukuoka, T. Yoshizawa, and N. Sakato, "Molecular mechanism of satratoxin-induced apoptosis in HL-60 cells: activation of caspase-8 and caspase-9 is involved in activation of caspase-3," Immunol Lett, vol. 84, pp. 23-7, 2002. [36] S. B. Bratton, M. MacFarlane, K. Cain, and G. M. Cohen, "Protein complexes activate distinct caspase cascades in death receptor and stress-induced apoptosis," Exp Cell Res, vol. 256, pp. 27-33, 2000.

Mohd Nazri bin Abu received his first tertiary qualification in Diploma of Medical Laboratory Technology from Universiti Teknologi MARA (UiTM), Malaysia in 2000. From 2001 to 2004, he was a Medical laboratory technologist at the PUSRAWI Hospital, Kuala Lumpur, Malaysia. In 2007 he earned his B.Sc (Hons) in Medical Technology from UiTM, Malaysia and then graduated from the Post Basic Cytopathology program from the Collage of Medical laboratory Technology, Ministry of Health, Malaysia, in 2009. Currently, he is pursuing a Ph.D degree in Health Sciences at the Universiti Teknologi MARA, Malaysia while working as a lecturer in the same university. He is a member of MIMLS (Malaysian Institute of Medical Laboratory Sciences). His research interest includes natural product, anti-diabetes, anti-cancer and cell signalling.

Muhammad Ashraf Mohd Salleh received his Bachelor of Science (Hons) in Microbiology from the Universiti Sains Malaysia (USM) in 2009. Upon graduation, he worked as a Field Science Officer at Sahabat Alam Malaysia, a non-government organization under Friends of Earth International until 2011. In early 2011, he started his study at Universiti Teknologi MARA (UiTM), Malaysia, at the Faculty of Pharmacy. He successfully converted his study from M.Sc to Ph.D level in June 2012.

Nabilatul H. Mohd-Radzman received her B. Sc. (Biotechnology) degree from Monash University in 2010. She worked as a research assistant at the Universiti Kebangsaan Malaysia (UKM) from 2010 to 2011 before joining the Faculty of Pharmacy, Universiti Teknologi MARA. Miss Mohd-Radzman is presently pursuing an M. Sc. degree at the same university. Her current research interests include the development of alternative therapies from natural products, and the cellular and molecular signalling in the development of metabolic syndromes like insulin resistance, diabetes and cancer. She received the Gold Medal Award at the 24th International Invention, Innovation & Technology Exhibition (ITEX) and a Travel Award from the 2013 Seoul International Congress of Endocrinology and Metabolism.

- 53 -

Journal of Medical Research and Development (JMRD)

Jul. 2013, Vol. 2 Iss. 3, PP. 48-54

Dr. Wan Iryani Wan Ismail received her BSc (Hons) and MSc from Universiti Sains Malaysia, Penang, Malaysia in Phytochemistry and Medical Microbiology. She then continued her doctoral study in Chemical Pathology (Cell Signalling) at the University of Cape Town, South Africa. She is currently a Senior Lecturer at the Faculty of Pharmacy, Universiti Teknologi MARA, and Malaysia. Her research interests are cell signalling and nutrient sensing in understanding molecular mechanism of anti-obesity, anti-diabetes and anti-cancer using natural products including honey and herbs. Several approaches have been used such as phytochemical analysis, protein expressions, metabolomics, enzyme assays, microscopic analysis and bioinformatics on bacteria, fungi, several types of cell culture and animal models to achieve the final goal i.e. understanding the molecular basis of obesity, diabetes and cancers, and their potent drugs in the future. She received the Gold Medal for Herb-based nutrition at the 24th International Invention, Innovation & Technology Exhibition (ITEX) in 2013. Dr. Ismail is the principal investigator for the study.

Dr. Rosmadi Mohd Yusoff received a Diploma in Microbiology from Universiti Teknologi MARA and a B. Sc. degree with Honours in Molecular Biology from the United Kingdom. He then continued to pursue a PhD in Microbiology from Universiti Kebangsaan Malaysia. He was a Research Assistant with Universiti Kebangsaan Malaysia from 2006 to 2007. He is currently a Senior Lecturer at the Faculty of Pharmacy, Universiti Teknologi MARA, and Puncak Alam, Malaysia. His research interests include molecular biology and genetic engineering, and more specifically, the gene expression analysis of lung cancer. Dr. Mohd Yusoff awards include the 2005 Bronze Medal for Prototype Lung Cancer Rapid Diagnostic Kit at Biotechnology Asia and the 2005 Silver Medal for Lung Cancer Diagnostic Kit at Eureka: 54th World Exhibition of Innovation, Research and New Technologies in Brussels, Belgium. Dr. Hamzah Fansuri Hassan is currently the Dean of the Faculty of Health Sciences, University Teknologi MARA, and Malaysia since 2010. He graduated from the State University of New York with a bachelor degree in biology in 1995 and received his master degree in the same field from the University of Bridgeport, Connecticut, USA in 1989. He earned his PhD in Anatomy from the Universiti Kebangsaan Malaysia in 2005. He is a member of the Counsel of Health Sciences Deans of the Malaysian Public Universities and is also a member of the Malaysian Radiation Physic Association. His current research interests are terahertz radiation modelling and tissue interactions, effect of natural products and cell signalling.

- 54 -