In Silico Analyses and Cytotoxicity Study of Asiaticoside and Asiatic Acid from Malaysian Plant as Potential mTOR Inhibitors.

Natural products remain a popular alternative treatment for many ailments in various countries. This study aimed to screen for potential mammalian target of rapamycin (mTOR) inhibitors from Malaysian natural substance, using the Natural Product Discovery database, and to determine the IC50 of the selected mTOR inhibitors against UMB1949 cell line. The crystallographic structure of the molecular target (mTOR) was obtained from Protein Data Bank, with Protein Data Bank (PDB) ID: 4DRI. Everolimus, an mTOR inhibitor, was used as a standard compound for the comparative analysis. Computational docking approach was performed, using AutoDock Vina (screening) and AutoDock 4.2.6 (analysis). Based on our analysis, asiaticoside and its derivative, asiatic acid, both from Centella asiatica, revealed optimum-binding affinities with mTOR that were comparable to our standard compound. The effect of asiaticoside and asiatic acid on mTOR inhibition was validated with UMB1949 cell line, and their IC50 values were 300 and 60 µM, respectively, compared to everolimus (29.5 µM). Interestingly, this is the first study of asiaticoside and asiatic acid against tuberous sclerosis complex (TSC) disease model by targeting mTOR. These results, coupled with our in silico findings, should prompt further studies, to clarify the mode of action, safety, and efficacy of these compounds as mTOR inhibitors.

Cytotoxic Effects of Betel Quid and Areca Nut Aqueous Extracts on Mouse Fibroblast, Human Mouth-Ordinary-Epithelium 1 and Human Oral Squamous Cell Carcinoma Cell Lines.

BACKGROUND: Betel quid chewing is more common among the older generation in rural areas of Malaysia. Oral cancer in Asia has been associated with the habit of chewing betel quid and areca nut. OBJECTIVE:   This study aims to investigate the cytotoxic effects of betel quid and areca nut extracts on the fibroblast (L929), mouth-ordinary-epithelium 1 (MOE1) and oral squamous cell carcinoma (HSC-2) cell lines. METHODS: L929, MOE1 and HSC-2 cells were treated with 0.1, 0.2 and 0.4 g/ml of betel quid and areca nut extracts for 24, 48 and 72 h. MTT assay was performed to assess the cell viability. RESULTS: Both extracts, regardless of concentration, significantly reduced the cell viability of L929 compared with the control (P<0.05). Cell viability of MOE1 was significantly enhanced by all betel quid concentrations compared with the control (P<0.05). By contrast, 0.4 g/ml of areca nut extract significantly reduced the cell viability of MOE1 at 48 and 72 h of incubation. Cell viability of HSC-2 was significantly lowered by all areca nut extracts, but 0.4 g/ml of betel quid significantly increased the cell viability of HSC-2 (P<0.05). CONCLUSION: Areca nut extract is cytotoxic to L929 and HSC-2, whereas the lower concentrations of areca nut extract significantly increased the cell viability of MOE1 compared to the higher concentration and control group. Although betel quid extract is cytotoxic to L929, the same effect is not observed in MOE1 and HSC-2 cell lines. Further investigations are needed to clarify the mechanism of action.
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Proliferation rate of stem cells derived from human dental pulp and identification of differentially expressed genes.

Stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs) obtained from the dental pulp of human extracted tooth were cultured and characterized to confirm that these were mesenchymal stem cells. The proliferation rate was assessed using AlamarBlue® cell assay. The differentially expressed genes in SHED and DPSCs were identified using the GeneFishing™ technique. The proliferation rate of SHED (P < 0.05) was significantly higher than DPSCs while SHED had a lower multiplication rate and shorter population doubling time (0.01429, 60.57 h) than DPSCs (0.00286, 472.43 h). Two bands were highly expressed in SHED and three bands in DPSCs. Sequencing analysis showed these to be TIMP metallopeptidase inhibitor 1 (TIMP1), and ribosomal protein s8, (RPS8) in SHED and collagen, type I, alpha 1, (COL1A1), follistatin-like 1 (FSTL1), lectin, galactoside-binding, soluble, 1, (LGALS1) in DPSCs. TIMP1 is involved in degradation of the extracellular matrix, cell proliferation and anti-apoptotic function and RPS8 is involved as a rate-limiting factor in translational regulation; COL1A1 is involved in the resistance and elasticity of the tissues; FSTL1 is an autoantigen associated with rheumatoid arthritis; LGALS1 is involved in cell growth, differentiation, adhesion, RNA processing, apoptosis and malignant transformation. This, along with further protein expression analysis, holds promise in tissue engineering and regenerative medicine.

In vitro cytotoxic evaluation of processed natural coral in human osteoblasts.

This study aimed to evaluate the in vitro cytotoxic effects of locally produced processed natural coral (PNC) using human osteoblasts (HOS). Cytotoxicity was not observed when HOS cells were cultured with PNC, as assessed by (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide; MTT) and Neutral Red (NR) assays at concentration up 200 mg/mL for up to 72 hours. Flow cytometry (FCM) analysis showed that PNC (200 mg/mL) did not decrease viability of HOS cells after 48 and 72 hours of treatment. In a cell attachment study, the HOS cells attached to the edge of the PNC disc, and later grew into the pores of the PNC disc. All results from these studies indicate that locally produced PNC material is noncytotoxic and favors the growth of HOS cells.

Evaluation of Tualang honey as a supplement to fetal bovine serum in cell culture.

The aim of this study was to evaluate Tualang honey as a supplement to fetal bovine serum in cell cultures using MTT assay, chromosome aberration test and gene expression analyses. The MTT assay showed the highest percentage of cell proliferation (105.3% increment than control) of human osteoblast cell line (CRL 1543) in 0.0195% honey in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. There was enhanced cell proliferation corresponding to the decrease in concentrations of honey as indicated by the mitotic index values when the osteoblast cell line was incubated at 37 degrees C for 48 hours. There were no chromosome aberrations both in the honey treated as well as distilled water treated (negative control) cell lines. In the case of gene expression analyses, fibroblast cell lines (CCL 171) were treated with honey (0.0195%) for 24 and 48 hours separately. Though there was over expression for the bcl-xl gene at both 24 and 48 hours, under expression for bcl-xs gene at 24 hours and over expression at 48 hours and under expression for both c-myc and p53 genes at both 24 and 48 hours, none of them were statistically significant in altering the expression of mRNA.