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Anticancer and apoptotic effects on cell proliferation of diosgenin isolated from Costus speciosus (Koen.) Sm
© Selim and Al Jaouni. 2015
Received: 2 April 2015
Accepted: 26 August 2015
Published: 2 September 2015
Diosgenin, a naturally occurring steroid saponin found abundantly in C. speciosus, is a well-known precursor of various synthetic steroidal drugs that are extensively used in the pharmaceutical industry.
The present study was conducted to evaluate the in vitro anticancer and apoptotic effects on cell proliferation of diosgenin isolated from C. speciosus (Koen.) Sm.
The results indicated that the treatment of HepG2 cells with the sample resulted in a cytotoxic effect as concluded from the IC50 value 32.62 μg/ml, while the treatment of HepG2 cells with paclitaxel, a known anti-cancer drug, resulted in an IC50 value of 0.48 μg/ml. The treatment of MCF-7 cells with the tested sample resulted in high inhibition in the cell viability, and resulted in an IC50 value of 11.03 μg/ml, while the treatment of MCF-7 cells with paclitaxel resulted in an IC50 value of 0.61 μg/ml. The levels of DR4 and caspase-3 were significantly increased (P < 0.01) in MCF-7 cells treated with the tested sample compared to untreated cells and possessed a similar activity of paclitaxel in DR4 induction but lower induction in caspase-3. On the other hand the treatment of macrophages or lymphocytes with diosgenin (250 μg/ml) resulted in an induction in the cell proliferation up to 3.2-fold and 2.1-fold of control, respectively.
The results presented here may suggest that diosgenin isolated from C. speciosus possess anticancer and apoptotic effects on cell proliferation, and therefore, can be used as pharmaceuticals drugs.
C. speciosus was procured from a fixed vendor in the local market of Al Jouf, KSA. Specimens were identified at the Aljouf University (Aljouf, Saudi Arabia) and voucher specimen (No. 13) was deposited at the Herbarium of the Department in the cited university. All cell maintaining materials was obtained from Cambrex, BioScience (Copenhagen, Denmark), while all other fine chemicals were from Sigma-Aldrich (CT, USA).
Hydrolysis and HPTLC analysis
Dried tuber powder (5 g) was treated with slightly modified method described by Drapeau et al. . Samples were hydrolyzed in 150 ml of refluxing 20 % H2SO4 in 70 % isopropanol for 8 h. The extract was filtered and extracted with hexane (50 ml × 3). The three hexane extracts were combined and rinsed thrice times with 5 % alkali and then rinsed thrice with distilled water. The extract was then passed through a column of Na2SO4 to eliminate any remaining water. The samples were concentrated to dryness by evaporating the solvent at 40 °C in a Rotary evaporator. The dried crude extract was solubilized in 0.5 ml of chloroform prior to the quantitative determination of diosgenin by thin layer chromatography as reported by Kshirsagar et al. .
Cell culture for cancer cell lines
Hepatocellular carcinoma Hep G2 Cells, breast adenocarcinoma MCF-7 cells and colon carcinoma HCT-116 cells were purchased from ATCC, VA, USA. HepG2 and MCF-7 cells were cultured in RPMI medium, while HCT-116 cells were cultured in McCoy’s medium. Both were supplemented with 10 % fetal bovine serum, 2 μmol/ml L-glutamine, 250 ng/ml fungizone, 100 units/ml penicillin G sodium, and 100 units/ml streptomycin sulphate at 37 °C in a humidified 5 % CO2 incubator. The cultures were passaged every four days by trypsinization using 1 ml trypsin/EDTA solution for 5 min at 37 °C. Cells were used when confluence had reached 75 %. Trypsin/EDTA solution consists of 0.25 mM trypsin and 1 mM EDTA dissolved in phosphate buffer .
Cell culture for immune cells
Lymphoblastic leukemia 1301 cells and raw murine macrophage (RAW 264.7) cell lines were purchased from ATCC, VA, USA. RAW 264.7 cells were grown in RPMI-1640. Media were supplemented with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, containing 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulphate, and 250 ng/ml amphotericin B. Cells were maintained at sub-confluency at 37 °C in humidified air containing 5 % CO2. For sub-culturing, monolayer cells were harvested by scraping. Cells were used when confluence had reached 75 % .
Preparation of cell lysates for ELISA
After cells treatment as the experimental design, collected cells were washed and centrifuged for 10 min at 1000 × g. Cell pellets were lysed in 0.5 ml of ice-cold lysis buffer. This buffer consists of 50 mM Tris HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5 % NP40, 1 % Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin (pH 7.4). Cell lysates were passed through a 21-gauge needle to break up cell aggregates, and then centrifuged at 14,000 × g for 15 min at 4 °C .
Cytotoxicity of tested sample against different cancer cell lines was measured using the MTT Cell Viability Assay. MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay is based on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium rings of the yellow MTT (5 mg/ml of MTT in 0.9 % NaCl) and form blue insoluble formazan crystals. Solubilization of the crystals by acidified isopropanol (0.04 N HCl in absolute isopropanol) was carried out. The extent of the reduction of MTT was quantified by measuring the absorbance at 570 nm . The number of viable cells is directly proportional to the level of soluble formazan dark blue color.
The half maximal inhibitory concentration of cell viability (IC50) was calculated by from the linear equation of sample concentration and cell viability %.
Cell death mode and mediators
Cell cycle analysis
MCF-7 cells (5×105) were collected after different treatments, and then washed two times with PBS, resuspended in 300 μl of PBS, and fixed with 4 ml of ice-cold 70 % ethanol. The cells were centrifuged to remove ethanol and washed once in PBS. The cell pellets were then resuspended in 1 ml of a solution of 0.1 % Triton X-100 in PBS, 0.2 mg/ml RNase A, and 10 μg/ml propidium iodide (PI) and incubated for 30 min at room temperature. The stained cells were analyzed by flow cytometry .
Fluorescence analysis of apoptosis and necrosis in living cells
Estimation of caspase-3 and death receptor-4
The levels of Caspase-3 and death receptor (DR-4) were measured in cell lysate by the quantitative indirect immunoassay ELISA technique, according to the method of Wang et al. . The following reagents were prepared: Washing buffer (PBS/0.05 % polyoxyethylene-20; Tween-20); blocking buffer (PBS/0.05 % Tween-20/5 % FBS); diluents buffer (PBS/Tween-20/1 % FBS); and substrate buffer (equal volumes of 3,3',5,5'-tetramethyl benzidine [TMB; o.4 g/]) and H2O2 [0.02 % in citric acid buffer; KPL, Kirkegaard and Perry Lab. Gaithersburg, MD, USA]). Briefly, 96-wells bottom polystyrene microplates were coated with cell lysates (50 μl/well) and incubated for 1 h at 37 °C and then washed by washing buffer before incubated with blocking buffer for 90 min at 37 °C. The wells were washed and then antibodies (1 mg/ml Rabbit polyclonal to DR-4 or caspase-3, Abcam Inc., Cambridge, MA, USA) were added. After the incubation for 1 h at 37 °C, polyclonal Goat anti-rabbit peroxidase conjugate (1:2000, Jackson Immunsearch Lab, USA) was added to wells. After 1 h, wells were washed and the substrate buffer was applied and the color development was stopped by adding 1 M HCl. The intensity of the developed yellow color was measured at 450 nm using microplate reader (FLUOstar OPTIMA, BMG LABTECH GmbH, Offenburg, Germany). The level of DR-4 was expressed as optical densities values and compared with the activity of paclitaxel.
Proliferation index of lymphocytes and macrophages
The effect of the tested sample proliferation index of lymphocytes and macrophages was investigated using the MTT Assay in lymphoblastic leukemia 1301 cells and raw murine macrophage (RAW 264.7). Cells (0.5×105 cells/well) in serum-free media were placed in a flat bottom 96-well microplate, and treated with 20 μl of different concentrations of the tested sample. After incubation for 72 h, cells were submitted to MTT assay. The results were compared with Echinacea purpurea root extract as a known standard immunostimulant.
Data were statistically analyzed using IBM computer supplied with Statistical Package for Social Scientists (SPSS) 10.00 for windows (SPSS Inc., Chicago, IL., USA). The student's unpaired t-test as was used to detect the statistical significance. P value was considered insignificant if more than 0.05.
Results and discussion
Currently chemotherapy is regarded as one of the most efficient cancer treatment approach. Although chemotherapy significantly improves symptoms and the quality of life of patients with liver cancer, only modest increase in survival rate can be achieved. Faced with palliative care, many cancer patients use alternative medicines, including herbal therapies. Conventional chemotherapy remains ineffective in curing hepatocellular carcinoma due to its high hepatotoxicity. Several plant-based extracts have been shown to be effective in liver cancer therapy and prevention . Given the several health promoting attributes to C. speciosus, the principle objective of the study was to evaluate and establish the anticancer efficacy of C. speciosus tuber extracts using human hepatocellular carcinoma cell lines. Previous work demonstrated in C. speciosus rhizome extract exhibited antioxidant and antiproliferative properties in human cancer cell lines . Our study represents the first time to study anticancer activity, apoptotic and inhibitory effects on cell proliferation of diosgenin isolated from C. speciosus.
Breast cancer is the most frequent cancer among women. Approximately 1.67 million new breast cancer cases were reported in 2012, which accounts for 25 % of all new diagnosed cancer . The current treatment strategies for breast cancer patients such as surgery, chemotherapy and radiotherapy or the combination of radiotherapy and chemotherapy have successfully increased the five-year survival rate of the breast cancer patients. Nevertheless, the long-term survival remains poor due to cancer relapse and metastasis. In the present study, diosgenin exhibited strong cytotoxic properties against caspase-3 deficient MCF-7 cells (Fig. 2c). According to the National Cancer Institute guidelines, diosgenin is a great candidate to be developed as an anticancer agent for breast cancer as the IC50 value was less than 11.03 μg/mL towards MCF-7 cells after treatment for 72 h . Diosgenin treatment on MCF-7 cell lines showed significant decrease in growth rate compared with control. On the other hand the percentage of non-viable cells on cell lines increased with the increasing period of treatment. These results were in concordance with the studies investigated the cytotoxic effect of Goniothalamin towards human breast cancer cells by Al- Qubaisi et al. . Hence present study shows the efficacy of diosgenin for the cytotoxicity towards MCF-7 cells thus suggesting protection against breast cancer.
Cell cycle analysis
Fluorescence analysis of apoptosis and necrosis in living cells
Estimation of caspase-3 and death receptor 4
Proliferation index of lymphocytes and macrophages
In conclusion, our current study is the first to identify the remarkable anticancer activity of, apoptotic and inhibitory effects on cell proliferation of diosgenin isolated from C. speciosus. Our data generate compelling evidence for further evaluation of this constituent as chemo preventive regimen for liver cancer. These studies are valuable for identifying plant-based chemotherapeutic drugs as currently used liver cancer drug causes many side effects. However, animal and human studies are indeed required to prove the safety and efficacy of application of diosgenin of C. speciosus as a potential chemo preventive agent in clinical practice.
This work was funded by Y.A. Jameel Scientific Chair of Prophetic Medical Applications, King Abdulaziz University, Kingdom of Saudi Arabia, under grant no. (PM 0008–1435). The authors, therefore, acknowledge with thanks Y.A. Jameel Scientific Chair of Prophetic Medical Applications technical and financial support.
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