Search of New Cytotoxic Crude Materials Against Human Oral Squamous Cell Carcinoma Using ¹H NMR-based Metabolomics

Materials and Methods

Materials. The following chemicals and reagents were obtained from the indicated companies: Dulbecco's modified Eagle medium (DMEM) (Gibco BRL, Grand Island, NY, USA); fetal bovine serum (FBS) (JRH Bioscience, Lenexa, KS, USA); 5-fluorouracil (5-FU) (Kyowa, Tokyo, Japan); dimethyl sulfoxide (DMSO) (Wako Pure Chem. Inc., Osaka, Japan); DMSO-d₆ (Cambridge Isotope Laboratories, Inc., MA, USA)

Plant materials. Crude plant extracts used in this study are listed in Table I. These extracts have been already prepared and employed for various screenings in our laboratory. All extracts were stored at −30°C before using. The original plants were mainly collected in various places in Japan. The specimens were proved and identified by Dr. Y. Shirataki and voucher specimens were also deposited in the Department of Pharmacognosy and Natural Medicines of Josai University.

NMR spectroscopy. ¹H NMR spectra were recorded at room temperature on a 400 MHz Agilent-400MR-vnmrs 400 spectrometer (Agilent Technologies, CA, USA). Each spectrum consisted of 65,536 complex data points and a spectral width of 6,410.3Hz, obtained by 16 scans with a repetition time of 5.0 sec and a relaxation delay of 1.50 sec per scan. The detection pulse flip angle was set at 45°.

NMR data reduction procedures and pattern recognition analysis. Each NMR spectrum was regimented into 375 regions, 0.04 part per million (ppm) wide, over the range −1.50 to 13.5 ppm. Each segment of the spectral regions (bucket) was integrated. Any integrated regions from 2.45 to 2.55 ppm that contained solvent signals were eliminated from the data table and then the total data were reduced to 372 regions. The remaining integral values of each spectrum were normalized over 100 total summed integrals to compensate for any differences in concentration between crude drug extracts. Spectral processing was performed using ALICE 2 for Metabolome version 5.0 software (JEOL Ltd., Akishima, Tokyo, Japan).

Preparation of crude drug extracts. Twenty-four kinds of crude plant extracts were prepared by extracting with MeOH under reflux for 3 h to give the methanolic extracts. These extracts were completely dried in vacuo and residues were dissolved in DMSO-d₆ at 10 mg/ml in concentration for 1H NMR measurement and dissolved in DMSO for cytotoxic evaluation.

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Figure 1.

The ¹H NMR spectra were subjected to principal component analysis (PCA). PCA score plot of PC1-PC2, in which 372 variables were equally accounted for in the data sets. The scores of PC1 and PC2 were 46.4, 31.8%, respectively. The circled dots show the sample exhibiting high TS value.

Cell culture. Human oral squamous cell carcinoma cell lines (HSC-2, HSC-4) were purchased from Riken Cell Bank, Tsukuba, Japan. Normal human oral cells, gingival fibroblast (HGF) and periodontal ligament fibroblast (HPLF), were prepared from periodontal tissues, according to the guideline of the Intramural Ethic Committee (No. A0808), after obtaining the informed consent from a 12-year-old patient at the Meikai University Hospital (9). Since normal oral cells have a limited lifespan of 43-47 population doubling levels (PDL) (9), the cells at 8-15 PDL were used in this study. All the cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate. The normal cells were detached by 0.25% trypsin-0.025% EDTA-2Na in phosphate-buffered saline (PBS) without Mg²⁺ and Ca²⁺ (PBS(−)) and subcultured at a 1: 4 split ratio once a week with a medium change in between the subcultures.

Assay for cytotoxic activity. The cells (3×10³ cells/well, 0.1 ml/well) were seeded in 96-microwell plates (Becton Dickinson and Company, Franklin Lakes, NJ, USA) and incubated for 48 h to allow cell attachment. Near-confluent cells were treated for 48 h with different concentrations of the test compounds in a fresh medium. The relative viable cell number of adherent cells was then determined by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (10). In brief, control and sample-treated cells were incubated for 4 h with 0.2 mg/ml of MTT in the culture medium. After removing the medium, the reaction product, formazan, was extracted with DMSO and the absorbance (the relative viable cell number) was measured at 540 nm by a microplate reader (Multiskan Bichromatic Labsystems, Helsinki, Finland). The 50% cytotoxic concentration (CC₅₀) was determined from the dose–response curve. The tumor specificity index (TS) was calculated by the following equation: TS=mean CC₅₀ (normal cells)/mean CC₅₀ (all tumor cell lines).