EVALUATION OF THE CYTOTOXIC EFFECT OF GINGER EXTRACT AGAINST PROSTATE CANCER MODEL USING IN VITRO STUDY

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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Umamaheswari et al. SJIF Impact Factor Volume 6, Issue 12, Research Article ISSN EVALUATION OF THE CYTOTOXIC EFFECT OF GINGER EXTRACT AGAINST PROSTATE CANCER MODEL USING IN VITRO STUDY Himayoon Rashid 1 and G. Umamaheswari* 1 Research Scholar, Research and PG Department of Biotechnology, Marudupandiyar College, Vallam- Post, Thanjavur, Tamilnadu, India Article Received on 06 October 2017, Revised on 26 October 2017, Accepted on 16 Nov DOI: /wjpps *Corresponding Author Dr. G. Umamaheswari Assistant Professor, Research and PG Department of Biotechnology, Marudupandiyar College, Vallam- Post, Thanjavur, Tamilnadu, India ABSTRACT Zingiber officinale (Ginger) is a potent source of various bioactive phenolics such as paradols, gingerols, shogaols and gingerones. The multiple benefits of ginger in alleviating inflammation, oxidative stress and proliferation promotes its use as a chemopreventive agent. Here we report the cytotoxic effect of bioactive fraction of ginger extract against prostate (DU145) cancer cell models and the underlying mechanism being involved using a in vitro strategy. The defined extract shows cytotoxic effect in dose dependent manner against cancer cell lines. Further, the ginger extract induced G2M cell cycle arrest in these models to the varying extent. Moreover, the bioactive fraction markedly reduced clonogenic capability of these cancer cells in dose escalating fashion. Lastly, the extract induced DNA fragmentation in these cancer model clearly showing that the cytotoxic effect is due to induction of apoptosis. This work supports the potential use of ginger as an chemotherapeutic agent for tackling distinct cancers. KEYWORDS: Ginger, Prostate cancer, DU145, Apoptosis, Clonogenic assay, DNA fragmentation assay. 1. INTRODUCTION Prostate cancer is the most common cancer in males and the second among cancers in the UK. Prostate cancer is related to androgen receptor signalling and studies have shown that attenuating this signalling ameliorates the prostate cancer progression. [1] Ginger across the Vol 6, Issue 12,

2 globe is used as a spice in foods and beverages and is quite known for its medicinal properties. Since time immemorial this rhizomatous perennial plant has been used as a remedy for treating different ailments like dyspepsia, gastritis, nausea and diarrhea. [2] Ginger has been reported to contain various phenolic compounds like paradols, shogaols, gingerols and gingerones. [3] Among the gingerols 6-gingerol is the most prominent and well study constituent of ginger. Regarding shogaols, 6-shogaol has maximum abundance. [2] The phenolic compounds present in ginger have shown antioxidant [4], anti-inflammatory [5] and anti-angiogenesis properties. [6] The constituents of ginger have been tested for their anticancer effect. Taking these grim facts into consideration we tried to explore the anticancer effect of bioactive fraction of ginger against prostate (DU145) cancer model using a sequential combinatorial in vitro strategy. We checked the cytotoxic effect of ginger extract against these cancer cell lines using MTT assay. In the downstream steps we investigated whether the defined extract induces cell cycle arrest in this cancer model. Further we tried to explore the ability of this extract to reduce the clonogenic ability of the defined cell lines. Finally we investigated the mechanism involved in the induction of cytotoxic effect on intervention with ginger extract. Our findings establish that ginger extract shows marked cytotoxic effect against these cell lines in dose dependent fashion, induces G2M arrest in these cells, reduces their clonogenic capacity. Our study showed that ginger extract provokes DNA fragmentation in these models culminating in apoptosis. 2. MATERIALS AND METHODS 2.1 MTT Assay This assay was used to see the cytotoxic effect of methanolic fractions of ginger on prostate cancer cell line (DU145) purchased from ATCC, USA. The assay is based on the capacity of mitochondrial dehydrogenase enzymes in living cells to convert the yellow water-soluble substrate 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) into a dark blue formazan product which is water insoluble. [7] Cells (5.0 X 10 4 cells) were plated in 96 well plates supplemented with DMEM and were incubated at 37 o C for 24 hrs. Plant extracts were tested at different concentrations (10, 20, 40, 80,160,320µg/ml) in serum free RPMI media and incubated for 24 hr in CO 2 incubator at 37 C. [8] After incubation with the different extract concentrations, the media was removed from the wells and 100 µl of the MTT reagent was added to each well and the plate was kept for incubation for 3-4 hrs. After incubation, the MTT reagent was removed before adding 100μL DMSO to each well and gently shaken. Vol 6, Issue 12,

3 Plant extracts treated cells were compared to untreated control wells. The absorbance at 570 nm using a microplate reader (ELx 800, Bio-Tek Instruments Inc., Winooski, VT, USA). 2.2 Fluorescence activated cell sorter (FACS) analysis 1 x 10 6 cells were plated in each well of 6 well plate supplemented with 2mL of DMEM media. After 24 hrs, cells were treated with or without 80 and 100 ug/ml extract. 1% DMSO was added to control wells and the plate was incubated for 24 hrs. The cells were pelleted down and the supernatant was discarded. Pellets were resuspended in 200 µl of 1X PBS and fixed overnight at in a 2 ml of fixing solution (70% ethanol). [9] Centrifuge was performed at 4000 rpm for 10 min at 4 C and the supernatant was again discarded. The pellets were washed twice with suitable amount of cold 1X PBS. This was followed by incubation for 15 min at room temperature in 500 ul of propidium iodide (PI) solution containing 0.05 mg/ml PI and 0.05 mg/ml RNase A in PBS. Finally the % of cells in various stages of cell cycle in compounds treated and un-treated populations were determined using FACS Caliber. [10] (BD Biosciences, San Jose, CA). 2.3 Clonogenic assay Cells (DU145) were plated 1 x 10 3 number of cells per 35mm dish and were kept for overnight incubation at 37 C. in CO 2 incubator. The cells were treated with methanolic fraction of Zingiber officinale extract at two different concentrations extract (160 and 320 μg/ml) keeping untreated cells as control. After hrs the media was removed and cells were incubated further. [11] The cells were incubated in 5% CO 2 incubator at 37 C for 1-3 weeks. Media was changed after 1 week and cells were further kept for incubation till colonies reached significant size and colonies having at least 50 cells were taken into consideration. After removing media, fixation was performed using 4% PFA for about 30 minutes followed by staining with crystal violet for about 1hr. [12] The excess crystal violet was washed with distilled water and the plates were allowed to dry. Colonies having more than 50 cells were counted using inverted microscope. Digital images were obtained using camera or scanning device. 2.4 DNA Fragmentation Assay This assay was performed for confirming the type of cell death. The apoptotic cells undergo DNA fragmentation which shows ladder life pattern in agarose gel. Cells (DU145) were seeded at a concentration of 106 per 35 mm dish incubated at 37 degree Celsius at 5% CO 2 for 24 h. The confluent cells were treated with different concentrations of extract for a period Vol 6, Issue 12,

4 % Inhibition Umamaheswari et al. of 24 hrs. The media was then aspirated and cells were washed with 1X PBS. After trypsinization, the cells were pelleted down by centrifugation. [13] Cell pellet was suspended in 0.5 ml lysis buffer (ph 7.8) [Tris-HCl 10 mm, ph 7.4; EDTA 10 mm, ph 8.0; Triton X % or Sodium-N-lauroyl sarcosinate], vortex vigorously and incubated at 4 C for 30 min. The lysate was centrifuged at 25,000 g for 20 min at 4 degree Celsius. [14] The supernatant was incubated with 20 g/l RNase A at 37 C for 1 h, then incubated with 20 g/l proteinase K at 37 C for 1 h leaving behind genomic DNA. [15] The DNA isolated from treated and untreated control was run on agarose gel and the images were captured using gel doc. 3. RESULTS AND DISCUSSION 3.1 Ginger extract shows cytotoxic effect against distinct cancers For evaluating the cytotoxic effect of methanolic fraction of ginger, DU145 cells were treated with different concentrations of the defined extract for 24 hrs. Our studies showed that ginger shows cytotoxic effect in the above mentioned cell lines in dose dependent manner. However, the effect was more intense in DU145 as evidenced from the IC 50 value of 45.3 µg/ml respectively (Figure 1) and Tables 1. These studies are in agreement with the previous findings demonstrating the cytotoxic effect of whole ginger extract against prostate cancer. [16] Thus it is quite evident that methanolic fraction of ginger provokes cytotoxic effect in prostate cancer models to a different extent and in dose dependent manner. 100 Cytotoxic effect of Zingiber officinale (Methanolic fraction) Concentration ( M) [logx] Figure 1. Cytotoxic effect of ginger extract (methanolic fraction on DU145 cell line). Vol 6, Issue 12,

5 Table 2. IC 50 of methanolic fraction of ginger extract against DU145 cells. Plants name Zingiber officinale (Methanolic fraction) Conc. OD at 590nm % mg/ml Trial 1 Trial 2 Trial 3 Mean Inhibition Control IC Ginger extract shows cytotoxic effect by causing cell cycle arrest As it is well established that certain drugs induce cytotoxic effect by causing cell cycle arrest thus we tried to evaluate whether similar is the case with the methanolic fraction of ginger. Our experimental studies showed that treatment of DU145 cells with the defined extract induced cell cycle arrest at G2M phase. While 10.71% and 34.99% arrest at G2M phase of cell cycle was seen in DU145 cells on treating with 80 and 100 µg/ml of ginger extract compared to untreated cells (18.05%), From these studies it is clear that methanolic extract of ginger induces cell cycle arrest at G2M phase of cell cycle in prostate cancer cell line to a varying extent (Figure 2A-D). Vol 6, Issue 12,

6 Figure 2A). Effect of DMSO (1%) on cell cycle arrest of DU145 cells. 2B). Effect of colchicine (20 µm) on cell cycle arrest of DU145 cells. Figure 2C). Induction of cell cycle arrest by methanolic fraction of ginger (80 µg/ml) in DU145 cell line. 2D). Cell cycle arrest caused by higher concentration (100 µg/ml) of ginger extract on DU145 cells. Vol 6, Issue 12,

7 HCT 16 DU 145 Umamaheswari et al. 3.3 Ginger extract restrains colony forming capacity of DU145 cancer cells Clonogenic assay is widely used for assessment of the capacity of cells to produce progeny. Cells which are reproductively viable form large colonies are said to be clonogenic while cells which have lost reproductive integrity cannot form such colonies. [16,17] Marked inhibition of colony forming capability was seen on DU145 cells on intervention with different concentrations of (160 and 320 µg/ml) of Zingiber officinale methanol fraction in comparison to untreated control. However maximum reduction in colony forming capacity was seen at higher dose (320 µg/ml) of Zingiber officinale. Nearly half the inhibition relative to control was seen at the concentration of (160 µg/ml). From these studies we summarize that ginger extract reduces clonogenic capacity of prostate cancer model in a dose escalating manner (Figures 3A). Methanolic fraction of Zingiber officinale (µg/ml) Control Methanolic fraction of Zingiber officinale (µg/ml) Control Figure 3A). Clonogenic assay performed with clones produced by DU145 tumor cells. Untreated control cells and Zingiber officinale plant extract treated (160 and 320 µg/ml) cells. Figure 3B. Percent survival of colonies after treatment of DU145 with different concentrations of Zingiber officinale. Reduction in survival followed dose dependent trend. Vol 6, Issue 12,

8 3.4 Ginger extract induces cytotoxic effect in cancer models by inducing apoptosis As the above studies point towards the cytotoxic effect to be apoptosis, we performed DNA fragmentation assay for validating our speculation. [13,14] The defined cell lines were treating with two different doses of ginger extract while the control plates were left untreated for 24 hrs. Ginger extract at both the doses induced apoptosis in the defined cell models as evidenced by the ladder like pattern of the DNA isolated from the treated plates compared to control plates (Figure 4). The banding pattern in treated samples resembled to that of a wells where the DNA ladder was run. Figure 4. DNA fragmentation after treatment of DU145 cells with increasing doses of ginger extract. DNA laddering was observed when DU145 cells were incubated with 160 and 320 µg/ml of Zingiber officinale extract for 20 h (Lane 1, 2 and 3 stands for control, 160 µg/ml and 320 µg/ml respectively Lanes 4 stands for 100 bp DNA Marker). CONCLUSION Our in vitro studies showed that the methanolic fraction of ginger induces cytotoxic effect in prostate cancer cell model in dose escalating fashion. Moreover, the defined extract provokes G2M cell cycle arrest to a varying extent in these models. Further, the ginger extract showed marked reduction in the reproductive integrity of DU145 cell line as evidenced by the significantly lesser number of colonies in treated plates in comparison to untreated plates. [16] However, the effect was found to be more drastic at the higher dose of ginger extract (320 µg/ml). The extract showed cytotoxic effect by inducing apoptosis in these cancer models as determined by the chromatin fragmentation or DNA laddering effect of treated samples. Speaking concisely the ginger extract exerts cytotoxic effect against prostate cancer model by Vol 6, Issue 12,

9 causing cell cycle arrest, inhibiting clonogenic ability and causing DNA fragmentation in these cells. Conflict of Interest The authors have no conflict of interest to declare. ACKNOWLEDGEMENT The authors wish to thank Assistant Professor G.Umamaheswari for professional assistance in the study and preparation of manuscript. REFERENCES 1. Gibbs A, Schwartzman J, Deng V, Alumkal J. Sulforaphane destabilizes the androgen receptor in prostate cancer cells by inactivating histone deacetylase 6. Proceedings of the National Academy of Sciences of the United States of America, 2009; 106: Zick SM, Djuric Z, Ruffin MT, Litzinger AJ, Normolle DP, Alrawi S, et al. Pharmacokinetics of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol and conjugate metabolites in healthy human subjects. Cancer epidemiology, biomarkers & prevention: a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 2008; 17: Shukla Y, Singh M. Cancer preventive properties of ginger: a brief review. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association, 2007; 45: Katiyar SK, Agarwal R, Mukhtar H. Inhibition of tumor promotion in SENCAR mouse skin by ethanol extract of Zingiber officinale rhizome. Cancer Res., 1996; 56: Surh YJ. Anti-tumor promoting potential of selected spice ingredients with antioxidative and anti-inflammatory activities: a short review. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association, 2002; 40: Kim EC, Min JK, Kim TY, Lee SJ, Yang HO, Han S, et al. [6]-Gingerol, a pungent ingredient of ginger, inhibits angiogenesis in vitro and in vivo. Biochem Biophys Res Commun, 2005; 335: Stockert JC, Blázquez-Castro A, Cañete M, Horobin RW, Villanueva Á. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. Acta Histochemica, 2012; 114: Vol 6, Issue 12,

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