Apoptosis Induction , Cell Cycle Arrest and in Vitro Anticancer Activity of Gonothalamin in a Cancer Cell Lines

Cancer is uncontrolled growth of cells. It can affect almost any part of the body. The growths often invade surrounding tissue and can metastasize to distant sites (WHO, 2011). Cancer is caused by mutations in the DNA. Normal cells repair the mutation or simply die when a mutation occurs whereas cancerous cells continue to survive with the mutations and they grow in an uncontrolled manner until a mass of cells known as tumor is created. Often the tumor interferes with the normal functioning of healthy tissues and can spread to other parts of the body (Tompa, 2007). Cervical cancer is a malignant neoplasm of the cervical area. It is an important women’s health problem in developing countries, killing 270,000 women each year. It is the third most common cancer overall and the leading cause of death from cancer among women in developing countries. At least 370,000 new cases are identified each year (WHO, 2010). Current cancer chemotherapy can damage or kill the rapid dividing and healthy cell but causes serious side effects such as nausea, anemia, and hair loss. In addition, the cost of chemotherapy drug is high as compared to the natural compound from medicinal plants. Goniothalamin, a natural occurring styryl-lactone and


Introduction
Cancer is uncontrolled growth of cells.It can affect almost any part of the body.The growths often invade surrounding tissue and can metastasize to distant sites (WHO, 2011).Cancer is caused by mutations in the DNA.Normal cells repair the mutation or simply die when a mutation occurs whereas cancerous cells continue to survive with the mutations and they grow in an uncontrolled manner until a mass of cells known as tumor is created.Often the tumor interferes with the normal functioning of healthy tissues and can spread to other parts of the body (Tompa, 2007).
Cervical cancer is a malignant neoplasm of the cervical area.It is an important women's health problem in developing countries, killing 270,000 women each year.It is the third most common cancer overall and the leading cause of death from cancer among women in developing countries.At least 370,000 new cases are identified each year (WHO, 2010).Current cancer chemotherapy can damage or kill the rapid dividing and healthy cell but causes serious side effects such as nausea, anemia, and hair loss.In addition, the cost of chemotherapy drug is high as compared to the natural compound from medicinal plants.
In this study, goniothalamin, a natural occurring styryl-lactone and extract from root of Goniothalamus macrophyllus is used to investigate the cytotoxic properties against several cancer cell lines.Furthermore, this study carried out to study the mechanism of apoptosis induction of goniothalamin on HeLa cells by determines the DNA content and Phosphatidyl Serine properties.

MTT Cytotoxicity Assay
All cell lines were trypsinized and counted using hemocytometer then were seeded in 96-well micro plate at 5×10 5 cells/ml and then incubated at 37 o C in 5%CO 2 to allow cells attachment.The medium was removed and replaced with fresh medium containing various concentrations of goniothalamin starting with the highest concentration of 60 µg /ml (two folded dilution).Cells were incubated at 37 o C, 5%CO 2 for 72 hours.Each concentration was assayed in triplicates (n=3).Seventytwo hours later, 20 µl of MTT (5 mg/ml) solution was added to each well and then the plate was further incubated for 4 h.All remaining supernatant were removed and 150 µl of DMSO was added to dissolve the formed crystal formazan.MTT assay reading was performed using ELISA plate reader (Tecan 200, USA).

The MTT Cell Proliferation Assay
To confirm anti-proliferative effects of goniothalamin on HeLa cells, MTT cell proliferation assay was carried out.In this assay, two different concentrations of compound with cells were prepared together with control.The concentration chosen were IC 25 and IC 50 concentrations (3.2 and 1.2 µg/ml).Each sample was assayed in triplicate, and control samples include cells without goniothalamin.The cells were treated by goniothalamin for 24, 48, and 72 hours.At the end of incubation periods, 20μl of MTT solution (5 mg/ml MTT dissolved in PBS) were added to each well containing cells and the plate was incubated at 37ºC in an atmosphere of 5%CO 2 for 4 hours.After that, most of the medium was removed, then a volume 100 µl of DMSO (dimethyl sulfoxide) was added into the wells to soluble the crystals.Finally the absorbance was measured by ELISA reader at a wavelength of 570 nm.Graphs (OD of samples against time) were plotted to determine the growth rates of cells in a given values.
Cells suspension was mixed with an equal volume of staining solution (1 : 1) containing 10 μg/mL acridine orange and 10 μg/mL propium iodide (dissolved in PBS) and observed under fluorescence microscope within 30 minutes.The viable (green intact cells), apoptotic (green shrinking cells with condensed of fragmented nucleus), and necrotic (red cells) were the morphological changes that were examined under fluorescence microscope.
HeLa cells were seeded in six-well plate and incubated at 37 o C in 5%CO 2 atmosphere.Twenty-four hours later, the medium in each well was removed and replaced with Goniothalamin at IC50 concentration dissolved in medium and incubated at 37 o C in 5%CO 2 atmosphere for 24, 48, and 72 h.After incubation period, Cells suspension was mixed with an equal volume of staining solution (1 : 1) containing 10 μg/mL acridine orange and 10 μg/ml propium iodide (dissolved in PBS) and observed under fluorescence microscope within 30 minutes.The viable (green intact cells), apoptotic (green shrinking cells with condensed of fragmented nucleus), and necrotic (red cells) were the morphological changes that were examined under fluorescence microscope (Leica, Germany).Each experiment was assayed three times (n=3) to provide a useful quantitative evaluation.Viable, apoptotic and necrotic cells was quantified in a population of 200 cells.The results were expressed as a proportion of the total number of the cells examined.

Analysis of Cellular DNA Content Using Propidium Iodide
HeLa cells at a concentration of 1x10 6 cells/ml were seeded into 6-well plate in 2 ml culture medium with a concentration of IC 50 value of goniothalamin and were incubated at 37ºC in an atmosphere of 5%CO 2 for 24,48 and 72 hours.Some wells were left with no treatment to be used as a control.After the incubation period, the cultured cells were harvested using trypsin and centrifuged.After incubation, the cells were detached and stained by using the Cycle TEST TM PLUS DNA Reagent Kit.Cell cycle was read using the Cell Quest software within 3 hours.
Flow cytometry (Annexin V/PI double staining): HeLa cells at a concentration of 1 X 10 6 cell/ml were seeded into the 6-well plate and treated with IC 50 concentration of Goniothalamin.After 24, 48 and 72 h incubation, the cells were detached and stained by using PE Annexin V Apoptosis Detection Kit I.All samples were read by the flow cytometer.

Statistical Analysis
Data was expressed as mean±SD.Statistical analysis was performed with Student's t-test using the independent t-test (SPSS version 15).Differences were considered significant at P=0.05.

The MTT Cell Proliferation Assay
The effect of goniothalamin on cells proliferation was studied in vitro, by using the MTT proliferation assay with HeLa cell lines.In the assay, both concentrations of GTN, IC 50 and IC 25 , were used.Untreated cells were used as control.To determine the changes in the numbers of cells in the wells during the experiment, cells proliferation had to be measured 24, 48 and 72 hours after the start of the incubation period.GTN treatment on HeLa cells showed that the optical density was lower in both concentrations, IC 50 and IC 25 , than controls.Whereas GTN treatment on HeLa cells with the IC 50 values showed that the optical density was lower than inoculation with the IC 25 values.This optical density is in proportion to the number of variable cells.Figure 1 shows that the growth rates decreased in the treated cells as compared with the untreated cells whereas inoculation with a higher concentration (IC 50 ) decreased the growth rate more than low concentration (IC 25 ).On the other hand, the percentage of non-viable cells treated with IC 25 value was 28% (day 1), 38% (day 2) and 45% (day 3).But the percentage of non-viable cells treated with IC50 values were 38% (day 1), 45% (day 2) and 48% (day 3) (Figure 2).

Acridine Orange (AO) and Propodium Iodide (PI) Double Staining using Fluorescent Microscopy
fluorescent microscope was conducted to study of morphological changes of cell death mode induced by goniothalamin after 24, 48 and 72 h.Acridine orange (AO) and propidium iodide (PI) staining was used.Viable cells displayed green fluorescence with the appearance of circular cell; intact DNA and nucleus give a round and green nuclei.The early apoptotic cells have fragmented DNA which gives several green colored nuclei and cell blebbing.Late apoptotic and necrotic cell's DNA would be fragmented and stained orange and red (Figure 3) Besides the study of morphological changes, the percentage of viable, apoptotic and necrotic cells also recorded in Table 2 and plotted as a graph in Figure 4.The percentage of apoptotic cells in untreated cells slightly increased from 0.67% after 24 h to 5.33% and 7% after 48 and 72h, respectively (Figure 5).Whereas, cells treated with goniothalamin at IC 50 concentration, the percentage of apoptotic cells increased rapidly from 37% after 24h to 53% and 63% after 48 and 72h, respectively.

Analysis of Cellular DNA Content Using Propidium Iodide
The DNA Content of HeLa cells were monitored by    Goniothalamin induced a significant time-dependent increase in the proportion of sub-G1 in HeLa cell population.However, a slight increase was observed at Sub G1 phase of untreated cervical cell (HeLa) over time.
Tables 3 and 4 show that the percentages of the treated cells in Sub-G1 increased from 5.91% at 24 hours to 27.99% at 48 h while the percentages in untreated cells increased from 2.44% at 24 hours to 6.62% at 48 h (p<0.005).
On the other hand DNA histogram showed that goniothalamin increased the population of cells at S phase in a time-dependent manner (Figure 6).The S population increased significantly from 6.17% and 8.53% in the untreated cells to 23.17% and 25.92% in cells treated with IC50 goniothalamin for 24 and 48h, respectively Tables 3  and 4. While concomitantly the G1 population decreased from 78.38 %and 69.27% in the untreated cells to 64.66% and 43.10% in the treated cells for 24 and 48h, respectively Tables 3 and 4. Similarly the G2/M population decreased from 12.98% and 15.58% in the untreated cells to 7.08% and 3.76% in the treated cells for 24 and 48h, respectively Tables 3 and 4. Flow cytometry (Annexin V/PI double staining) Apoptotic cells exclude all dyes which are in use for cell viability assays, such as PI, while necrotic cells do not.In cells with a damaged cell membrane PI induces a red fluorescence on the DNA, whilst it is excluded by cells with a preserved cytoplasm membrane.Hence during the initial phase of apoptosis, the cells are still able to exclude PI and therefore do not show any red fluorescence signal, similar to that of living cells.Figure 7 showed the results of Annexin V/PI flow cytometry of HeLa cells after treatment with IC 50 value of goniothalamin.Untreated cell was found in the lower left quadrant of the cytograms, these viable cells excluded PI and were negative for Annexin V binding.The upper right quadrant represents the nonviable, necrotic cells, positive for Annexin V binding and showing PI uptake.The lower right quadrant represents the apoptotic cells, Annexin V positive and PI negative, demonstrating Annexin V binding and cytoplasmic membrane integrity (Figure. 11).The Annexin V/PIapoptotic cell population for HeLa cell line increased from 6.4%in untreated cells, to 26.45% in treated cells at 24 h post-infection.

Discussion
Cell death in mammalian cells are divided into two morphologically and biochemically distinct modes namely apoptosis and necrosis (Doyle and Griffiths, 1998).Apoptosis is an organized, pre-programmed response of cell to shifting of environmental conditions.Characteristics of apoptotic cell include cell shrinkage, nuclear and DNA fragmentation and breaking up of the cell into membrane-bounded vesicles, termed 'apoptotic bodies', which are subsequently ingested by macrophages (Doyle and Griffiths, 1998).Apoptosis plays a vital role in regulating growth, development and immune response, and also clearing abnormal cells (Fan et al., 2005).This apoptosis program becomes important in medical study in order to cure the cancerous cell without give the inflammatory effect.Aberrant cell death processes may underlie many human diseases including cancers, autoimmune, neurodegenerative and immunodeficiency disorders (Baehrecke, 2002).
Cytotoxic has been defined as the cell killing property of a chemical compound independent from the mechanism of death (Graham-Evans et al., 2003).Cytotoxicity assay is an appropriate method for screening new substances within a short time in order to determine cytotoxicity on cancer cells (Alley et al., 1988).The effective dose for a 50% reduction in cell number for plants products to be considered cytotoxic should be less than 20 µg/ml (Geran et al., 1972 ).
In this study, GTN have indicated significant growth inhibition in HeLa cell line at low concentration of IC 50 values.MTT proliferation assay was carried out to determine the growth rate of cells.A linear relationship between the formazan generated and the number of viable cells was demonstrated, together with time-dependent growth characteristics for HeLa cells (Ferrari et al., 1990).GTN treatment on HeLa cells cell lines showed significant decrease in growth rate compared with control.Whereas treatment with high concentration (IC 50 value) showed that the growth rates of the cells were more decreased than of low concentration (IC 25 values).On the other hand the percentage of non-viable cells on both cell lines increased with the increasing period of treatment.
However, MTT cytotoxic results of GTN on HeLa cells have been further supported with morpgological study using fluorescent microscopy Acridine Orange Propidium Iodide staining assay and flow cytometric analysis of cell cycle.The apoptotic features were confirmed and the percentage of apoptotic cells was determined from at least 300 counted cells observed under fluorescent microscope.The calculation of apoptotic cells is described as the percentage of apoptotic cells and apoptotic bodies within the overall population of cells.The percentage of apoptotic cells and the graph showed that the percentage of apoptotic cells treated with goniothalamin was increasing among the time.These distinctive morphological features form the basis of some of the most widely used techniques for the identification and quantification of apoptosis, and thus morphologic description using Phase Contrast microscopy and fluorescence microscopy remains one of the best ways to define apoptosis (Doonan and Cotter , 2008).
The quantitative analysis of cell cycle is very important in the study of molecular mechanism of cell death and cell cycle progression (Tao et al., 2004).Untreated and treated HeLa cells were evaluated for apoptosis by measuring the amount of apoptotic cells using of DNA flow cytometry (FCM).Flow cytometric analysis of cell cycle measures the apoptotic changes in cells by staining them with DNA dyes (Telford et al., 1994).Apoptotic cells, due to a change in membrane permeability, showed an increased up-take of the vital dye, PI, compared to live cells (Nicoletti et al., 1991;Telford et al., 1994).This method is useful for quantitative estimates of the fractions of cells in the different phases of the cell cycle (Ali et al., 2011).In this study goniothalamin treatment on HeLa cells produced S phase cell cycle accumulation with a large increase in the sub-G1 which mean there was a relationship between goniothalamin-induced S phase arrest and apoptosis(p<0.001).A study of cell cycle pattern has been documented that goniothalamin treatment causes cell cycle arrest and cell death maximally at G2/M phase (Chen et al., 2005).Another study demonstrates that GTN arrested cell cycle at G0/G1 in SK-Hep1, and at G2/M in Hep-3B cells (Cheng-Hui , 2008).These results concurred with the previous results to suggest that goniothalamin induce apoptosis on HeLa cells more extensively with increasing in time.
Change in plasma membranes is the earliest features of apoptosis.In apoptotic cells, the membrane phospholopid, phosphotidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane thereby exposing PS to the external cellular activity (Lawen, 2003).Annexin binding assay is a method permits the detection of the early phases of apoptosis before the loss of cell membrane integrity (Vermes et al., 1995;Aubry et al., 1999).The principle of Annexin V staining method used is the conjugation of Annexin V to phosphotidylserine of the apoptosis cells and in conjunction of dye Propodium Iodide which binds to cells at different stage and distinguishes apoptosis cells with necrotic cells (Tao et al., 2004).Apparently, the results indicate that the percentage of cells in early apoptosis of the cervical cancer cell (HeLa) treated with Goniothalamin appeared after 24 hr.
The percentage of the cells treated with Goniothalamin were decreased in early apoptosis phase and increased in late apoptosis over time.
In summary, goniothalamin (GTN) showed selective cytotoxic towards cervical cancer (HeLa), breast carcinoma (MCF-7), and colon cancer (HT29) but is not normal mouse fibroblast cells (3T3).The compound is potentially a good anti-cancer drug since it is non-toxic towards healthy cells.Our results indicate that GTN inhibits HeLa cell proliferation via apoptosis and causes cell cycle arrest and cell death at S phase.

FigureFigure 2 .
Figure 1.MTT Proliferation Assay for IC 50 and IC 25 Goniothalamin Concentrations (3.2 and 1.2 µg/ml) Against HeLa Cells at 24, 48 and 72 Hours Post-Treatment.The growth rates decreased in the treated cells as compared with the untreated cells whereas inoculation with a higher concentration of virus (IC 50 ) decreased the growth rate more than low concentration (IC 25 )

Figure 4 .
Figure 4. Flourecent Microscopy Examination.Percentage of apoptotic cells, necrotic cells and viable cells in HeLa cell population with gonithalamin treatment after 24 48 and 72 h.HeLa cell death via apoptosis increased significantly (*P < 0.05) in time-dependent manner

Figure 5 .Figure 6 .Figure 7 .
Figure 5. Cell Cycle Analysis of Cervical Cell Cancer Treated with Goniothalamin at IC 50 Concentration.A) Untreated cells, B) Treatment after 24h, C) Treatment after 48 h.

Table 4 . Cell Cycle Analysis of Cervical Cancer Cells (HeLa) at 24h and 48h Treated with Goniothalamin
flow cytometry after propidium iodide staining nuclei.