Anti Tumor Effect Of Ginsenosides On Nasopharyngeal Carcinoma Biology Essay

Published: Last Edited:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Nasopharyngeal carcinoma (NPC) is a cancer located at the upper part of pharynx where the nasal cavity and auditory tube meets. Chemotherapy is one of the treatment method for NPC patients using conventional anti-cancer drugs such as cisplatin and paclitaxel. Among the thousand types of Traditional Chinese medicine, ginseng has been used for anti-cancer treatment for many years and its effect has been found to attribute to the pharmaceutical active compound - ginsenosides. In this project, the anti-cancer effect of conventional anti-cancer drug including Docetaxel, Paclitaxel, Colchicine, Vinblastine, Nocodazole and Cisplatin (0.01 pM - 10 μM) and ginsenosides PPD, PD, S-Rh2, RP-1, CK, R-Rh2, R-Rg3 and S-Rg3 (1 μM - 100 μM ) are tested. Our data showed that, 13 out of 14 screened compounds exhibited cytotoxicity effect on NPC HK-1 cells in a dose-dependent manner. By comparing their IC50 values, the order of cytotoxicity of conventional drugs is Vinblastine > Colchicine > Nocodazole > Docetaxel > Paclitaxel > Cisplatin. While the order of cytotoxicity of ginsenosides is PD > PPD > CK > S-Rh2 > RP-1 > R-Rh2 > R-Rg3 > S-Rg3. Among them, PD and Vinblastine are the most potent candidates. The increase of sub-G1 phase in drug-treated cells were studied using flow cytometry assay. The result indicated that the cytotoxic effect is not due to the induction of apoptosis but the G2 phase cell cycle arrest.

1. Introduction

1.1 Nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a cancer located at the upper part of pharynx where the nasal cavity and auditory tube meets. NPC is a cancer uncommon in most of the world but very common in South East Asia (Jemal et al., 2011). The environmental and eating habit have been suspected to be a cause of the cancer as well as the family inheritance. But it is the invasion of Epstein-Barr virus (EBV) that is the top reason for nasopharyngeal carcinoma. EBV is one of the most common virus infecting over 90% of adult (David et al., 2004). Over 90% of eight-year-old children in Hong Kong have been infected by EBV (Kangro et al., 1994). Although the infection is life-long, EBV will not cause harm to human unless the balance between human and virus changed. Epstein-Barr virus is potentially tumorigenic due to the presence of membrane proteins LMP1, which is the oncogene of nasopharyngeal carcinoma (Plaza et al., 2003).

The symptons of NPC are blood-stained nasal draining, nasal obstruction, blocked feeling in ear, facial pain and headache. It is difficult for people to discover the disease not untill the final stage of cancer because the symptons in early stage of NPC are common and people usually under-estimate them. According to the Hong Kong Cancer Registry, Hospital Authority, 2008, NPC ranks the 7th in the top 10 most common cancers for both men and women. There are now several methods to treat nasopharyngeal carcinoma including surgery, radiotherapy and chemotherapy. Radiotherapy is the most common treatment but it can only treat primary tumor and it cannot prevent the cancer cells from undergoing metastases. Chemotherapy is a treatment that comes along with radiotherapy. Surgery is the least common and recommanded method for treating NPC. Not only the location of cancer is quite complicated and difficult to reach for surgery, it is not good looking for someone to have a large scar on his/her face.

It is a common fact that prevention is always better than cure. Though the genetic inheritance and infection of Epstein-Barr virus are something that we cannot prevent, people can attain a healthy life style for NPC prvention, such as avoiding smoking, drinking alcohol and visit the dentist regularly.

Figure 1. Location of nasopharyngeal carcinoma (NPC)

1.2 Ginseng

Ginseng, root of Panax genus in the family Araliaceae, is mostly found in China and Korea. The Chinese have been using ginseng as a medical herb for thousands of years. They used the name ginseng (Jin-chen in Chinese) because of the meaning "Man Plant" which stems from its frequent likeness to the human form. It was later given the genus name "Panax" meaning "all heal" in Greek. Currently, there are four forms of Panax Ginseng that are sold in the market - the fresh ginseng, the red ginseng (RG), the white ginseng (WG) and the sun ginseng (SG). Fresh ginseng is the raw product that harvested from the gound. Red ginseng is steamed form of fresh ginseng, it's biochemical composition is althered to be a better medical herb. The white ginseng is peeled and then dried to reduce water content. It's yellowish-white color is caused by the bleaching effect of drying in sun. The sun ginseng is further heated the red ginseng to increase the amount of active compound of ginseng.

Ginseng is known as adaptogen that can inprove human immune system, increase physical and mental endurance, enhance sexual desire, combat fatigue and stress, strengthen and restore human energy. It has been proved to have various biologcal effects including anti-aging, anti-cancer and reducing depression (Yue et al., 2007). Due to its multi-functional aspects, ginseng has been widely used in pharmaceutical products, nutritional food and cosmetic products. However, not everybody can benefit from intaking ginseng, pregnant or nursing women or children should avoid ginseng as it may lower estrogen production.


Ginsenosides, a kind of saponins and are active compounds of Panax ginseng. There are over 30 ginsenosides that have been discovered and they can be mainly divided into two groups: protopanaxadiols (PPD) (eg. Rb1, Rb2, Rc, Rg3 and Rh2 ) (Figure 2) and protopanaxatriols (PPT) (eg.Rg1, Re, Rf and Rh1) (Figure 3) (Wang et al., 1997). They are differed by the number of attached sugars and the attachment sites of hydroxyl group (OH). Ginsenosides are actually biotransformer, they can be easily converted into another ginsenoside by loosing or gaining a hydroxyl group or sugar. For example, Rg3 contains two glucoses at R1, it becomes Rh2 when one glocuse is lost. But when a glucose is added to Rh2 at the site of R3, it turns into F2 (Figure 4). However, these two groups of ginsenosides possess very different biological property. They are said to possess Yin/Yang actions. For example, PPD inhibits angiogenesis but PPT promotes angiogenesis (Yue et al., 2007).

Figure 2 Structure of protopanaxadiols (PPD)

Figure 3 Structure of protopanaxatriols (PPT)

Figure 4 Chemical structure of ginsenosides (Yue et al., 2007)

1.4 Application of ginsenosides as anti-cancer drug

Although many conventional drugs has been developed to treat cancer, they often induce side effects. Docetaxel often causes alopecia, neutropenia and anaemia (Lyseng and Fenton, 2005). Cisplatin often induces nausea and vomiting on patient. (Cooley et al., 1994). There are many researches have shown that ginsenosides possess anti-cancer effect and can enhance the effect of other chemotherapeutic drugs. For example, ginsenoside Rh2 was found to increase the effect of docetaxel in treating prostate cancer (Musende et al., 2010). PPD likes Rg3 also proved to have anticancer activities on human breast cacner cell MCF-7 (Ha et al., 2010). Compound K is able to induce apoptosis of human leukemia cells (Cho et al., 2009). Since certain conventional drugs has been developed to treat cancer including NPC, I would like to study if cotreating conventional drugs with ginsenosides can improve the anti-proliferation effect of conventional drugs on NPC. ie, to reduce the concentration used by current drugs as they usually cause side effects to the patient. For example, according to the US Food and Drug Administraion, the current concentration used in pharmaceutical of Cisplatin and Docetaxel are 1mg/ml and 10 mg/ml respectively.

1.5 Characteristics of conventional drugs

1.5.1 Characteristics of Docetaxel

Docetaxel is a semi-synthetic chemical extracted from European yew tree (Clarke et al., 1999). Docetaxel works as an atimicrotubule drugs by promoting and stabilising microtubule assembly which leads to insignificant free tubulin for microtubule formation. This in turn interfere microtubule depolymerisation and as a result, normal cell division is inhibited (Yvon et al., 1999). Docetaxel is a well established chemotherapy drug because of its antineoplastic activity which make it against a wide range of cancer cells like breast and ovarian cancer (Lyseng and Fenton, 2005).

Since docetaxel is an atimicrotubule drugs, it specifically work on cell cycle and toxic to all dividing cells such as hair follicles, bone marrow and other germ cells (Rang et al., 2003). It will lead to common chemotherapy side effects such as alopecia, neutropenia and anaemia.

Figure 5. Structure of Docetaxel

1.5.2 Characteristics of Paclitaxel

Paclitaxel is a mitotic inhibitor which used in treating cancer during chemotherapy. Same as docetaxel, paclitaxel is isolated from the Pacific yew tree, Taxus brevifolia and is named as taxol (Goodman et al., 2001). Paclitaxel expresses its effect in the same way as Docetaxel does, to stabilizes the microtubule polymer and prevent them from separaton. In this way, chromosomes cannot achieve a metaphase spindle configuration and will lead to a mitotic block (Jordan and Wilson, 2004), which will eventually cause cell apoptosis or switch the cell back into the G1-phase without cell division (Brito et al., 2008).

Same as other chemotherapy drugs, Paclitaxel will cause side effects including vomiting, nausea and, loss of appetite. There is research that showed Paclitaxel can damage ovarian in rats and lead to infertility (Ozcelik et al., 2010).

Figure 6. Structure of Paclitaxel

1.5.3 Characteristics of Cisplatin

Cisplatin is a compound of class of platinum-containing chemotheraphy drugs. carboplatin and oxaliplatin are also members of the class. cisplatin was first developed by Alfred Werner in 1893. Cisplatin is a anti-cancer drug because of its ability to bind to and cause crosslinking of DNA. The damaged DNA will trigger DNA repair system, however, if repairment cannot help, the cell will eventually trigger apoptosis (Trzaska, 2005). Cisplatin is actually a stereoisomer . It has the trans- form name transplatin. However, Transplatin does not interact with DNA in the same way as cisplatin does. trans-cisplatin is not an effective chemotherapeutic agent as it will rapidly degraded before reacting with DNA (Woollins et al., 1983).

As usual, Cisplatin will elict bad side effect while treating cancer. For example, joint pain, ringing in the ears and hearing trouble. A way to minimize side effects is to lower therapeutic doses use (Trzaska, 2005).

Figure 7. Structure of Cisplatin

1.5.4 Characteristics of Vinblastine

Vinblastine is a microtubule inhibitor which was first separated from the Madagascar periwinkle plant and its cell-killing ability has been discovered by Robert Noble and Charles Beer since 1950s (Duffin, 2000). Vinblastine kills cancer cell by binding to the tubulin which in turn stop the assembly of microtubules. Vinblastine is found to work by two mechanisms, suppress microtubule at low concentration and reduce microtubule polymer mass at higher concentration (Jordan and Wilson, 2004). Due to the above ability, Vinblastine now acts as a first-line chemotheraphy drug in treating lung cancer , neck cancer and Hodgkin's lymphoma (Canellos et al., 2003).

Figure 8. Structure of Vinblastine

1.5.5 Characteristics of Nocodazole

Microtubules play an important role in controlling the move of actin as well as the migration of chromosomes which is a crtitical step in cell division. Chemicals that can affect the microtubule from normal functioning can cause cell death (Schwartz, 2009). Nocodazole is another microtubule-targeting drug. It binds to tubulin to induce microtubule depolymerization (Chang et al., 2008) and hence the cell loss the ability to trigger actin activity. When chromosome cannot migrate properly, cell locomotion is prevented (Schliwa et al., 1999). Once the cancer cell cycle is stopped in metaphase (Yan et al., 2009), it cannot ungergo cell division and the cancer can be controlled.

Figure 9. Structure of Nocodazole

1.5.6 Characteristics of Colchicine

Colchicine, a microtubule targeted chemotheraphy drug, can bind to tubulin and break down microtubules into filament in an irreversible manner (Wisniewski et al., 1968). Tubulin is a critical substance for cell mitosis, and so colchicine acts as a mitotic poisoner or spindle poisoner to inhibit cell division and promote cell death***. Colchicine is also a strong neurotoxin which affects the brain memory (Meyers et al., 1997).

Colchicine, like other chemotherapy drugs, will cause side effects including gastrointestinal upset and neutropenia. It can even damage bone marrow and lead to anemia when high dose is applied.

Figure 10. Structure of Colchicine

2. Materials and Method

2.1 Chemicals and regents

Ginsenosides CK, RP-1, S-Rh2, R-Rh2, PD, PPD, S-Rg3 and R-Rg3 were purchased from Sigma-Aldrich Chemical Co. Except for PD which was dissolved in ethanol in stock solution, others were dissolved in DMSO. The conventional anti-cancer drugs Docetaxel, Paclitaxel, Colchicine, Vinblastine, Nocodazole and Cisplatin were also obtained from Sigma-Aldrich Chemical Co.

2.2 Cell Culture

HK-1 cells are cultured in RPMI 1640 medium with 2.0 g/l sodium bicarbonate plus 10% fetal bovine serum (FBS) at 37°C incubator containing 5% CO2.

2.3 Drug Treatment

2.3.1 Treatment of HK-1 cells with ginsenosides and western drugs

Ginsenosides or conventional drugs were added to the HK-1 cells which were seeded in a 96-well plate with density of 8 x103 cell per well after one day of incubation at 37°C. The treatment was then further incubated at 37°C for 48 hours.

2.3.2 Cotreatment of ginsenosides with western drugs

HK-1 was seeded in 96 well plate at a density of 8 x103 cells per well. Same amount of ginsenosides and western drugs of selected concentration were added to the HK-1 cells at the same time. The HK-1 cells were then incubated for 48 hours treatment.

2.4 Cell Viability Assay

HK-1 cells were plated at a density of 8 x103 cell per well in a 96-well plate with 100 µl RPMI 1640 in 10% FBS. Cells were allowed to grow for 24 hours and then different ginsenosides and western durgs were treated. After 48 hours of treatment in 37°C incubator, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) (MTT), a substance that will be reduced to purple formazan in living cells (Twentyman and Luscombe, 1987), were added to each well and further incubated for 3 hours. Lastly, 100µl of dimethyl sulfoxide (DMSO) was added to dissolve the purple formazan crystal into a violet colored solution and the absorbance of the solution was measured at 540 and 690 nm by a spectrophotometer (Tecan infinite 200 PRO).

2.5 Degree of interaction analysis

After the cell viability was found by MTT assay, the data was also analysed by the Calcusyn software program (Biosoft, MO 63135, USA). The calculated Combination Index (CI) is a quantative measure of the degree of interaction between different drugs. If CI values line between 0.1 to 0.3, it indicates strong synergism. If CI vlaue is between 0.3 to 0.7, it represents synergism. CI value of 0.7 to 0.9, slightly synergism; CI value ranges from 0.9 to 1.1, nearly additive. And if CI value is larger than 1.1, antagoism (Morelli et al., 2005).

2.6 Flow Cytometry Assay

HK-1 cells were plated at a density of 3 x105 cell per well in a 6-well plate with 2 ml RPMI 1640 in 10% FBS. Cells were allowed to grow for 24 hours and then 50 µM of S-Rh2 and R-Rh2 with western durgs were treated. After treating in 37°C incubator for 48 hours, both adherent and detached cells were harvested. Cell pellet was washed with 2 ml PBS, and then either fixed with 75% ethonal at 4°C refrigerator for 1 hours or at -20°C refrigerator for overnight. Lastly, cells were mixed with ribonuclease and stained by propidium iodide solution for half an hour in dark and then DNA content was analyed by FACScan flow cytometer (Morelli et al., 2005) and calculated the gated region on the DNA content histogram by Cell Quest software. The cell cycle data analysis was done by the MOOD Fit program.

Figure 11. Mechanism of flow cytomotry

3. Results

3.1 Effect of Ginsenoides on HK-1 Cell Viability

Eight different ginsenosides were added to HK-1 cells for 48 hours to study their effects on cell viability. PD (1 µM to 60 µM) was found to be the most effective ginsenoside on killing HK-1 as it had the smallest IC50 values of 25 µM. PPD (1 µM to 60 µM) and CK (60 µM to 80 µM) had similar effect on HK-1 cells with IC50 value of 40µM and 45µM respectively (Figure 13). For S-Rh2 and RP-1, they both can kill almost all HK-1 cell at high dosage but they cannot kill half of the cells' population untill 60 µM. The R-Rh2 (40 µM to 80 µM) and R-Rg3 (1 µM to 100 µM) did not have much effects on HK-1 cells. They could only killed around 10 to 20 % of the cells even at the highest concentraions of 100 µM. Lastly, S-Rg3 showed very little effect on HK-1 cell growth from 1 µM to 10 µM. However, when high dosgae was applied, the cell viability of HK-1 cells increased. There was 50% increasment in cell growth when 75 µM of S-Rg3 was added. Overall, all the tested ginsenosides except S-Rg3 were found to have some positive effects on cell apoptosis and the effect was dose-depended. It was the S-Rg3 that caused cell death at low concentration (10 µM) but promote cell growth at high concentration (100 µM).







Figure 12. Effect of ginsenosides A) S-Rh2 and R-Rh2, B) S-Rg3 and R-Rg3 , C) PPD, D) PD, E) RP-1 and F) CK on cell viability on HK-1 cell by MTT assay. HK-1 were cultured and treated with different ginsenosides for 48 hours. The extent of cell death was determined and expressed as a percentage of that with untreated control. Data are means ± SD of three independent experiments.

Figure 13. Comparison of IC50 values of various ginsenosides with different testing concentration range base on Figure 13. R-Rh2, S-Rg3 and R-Rg3 are not shown above as their cytotoxic effects do not reach IC50 level.

3.2 Effect of Western drugs on HK-1 Cell Viability

Docetaxel, Paclitaxel, Colchicine, Vinblastine, Nocodazole and Cisplatin are conventional anti-cancer medication. They are now mainly used for treatment of breast (Frances et al., 2011) and lung cancer (Gadgeel, 2011). MTT assay was used to find their dose response curves toward nasopharyngeal carcinoma HK-1 cell. The testing dosage was from 0.01 pM to 10 µM. We can see that from Figure 5, all of the six drugs showed obvious positive effect on inhibiting cell growth. Among which, Vinblastine has the greatest effect on HK-1 cells with the smallest IC50 value (0.05 μM). Colchicine is the second strongest with IC50 value of 0.07 μM. Nocodazole could also kill 50% of cells at about 0.1 μM. Docetaxel and Paclitaxel have similar effect on HK-1 cell viability with 40% of cells death at 10 µM. Cisplatin is the weakest one that could only cause 10% cells death at the highest testing concentration.

Figure 14. Effect of six drugs on cell viability of HK-1 cell by MTT assay. HK-1 were cultured and treated with six drugs respectively (0.01pM, 0.1pM, 1pM, 0.01nM, 0.1nM, 1nM, 0.01µM, 0.1µM, 1 µM and 10 µM) for 48 hours. The extent of cell death was determined and expressed as a percentage of the untreated control. Data are means ± SD of four independent experiments.

3.3 Effect of cotreatment of Ginsenoside S-Rh2 and six Western Drugs on HK-1 Cell Viability

Upon finding the dose response curves of various ginsenosides and western drugs, S-Rh2 (50 µM) was selected for cotreatment with the six conventional drugs (0.1 pM to 0.10 µM) to see if there is any synergistic effect on killing nasopharyngeal carcinoma cells. The results show that S-Rh2 do not have synergistic effect with most of the conventional anti-cancer as expected. The result of cotreatment of S-Rh2 and Vinblastine was the worst. The cotreatment of 50 µM of S-Rh2 and 0.1 pm to 0.1 nM of Vinblastine did not have further drop in cell viability at all. S-Rh2 could further reduce the cell viability with Docetaxel by 10%. For Paclitaxel, there is significant drop in cell viability at 0.1 pM and 1 pM of Paclitaxel and 50 µM of S-Rh2 than single treated with Paclitaxel. However, the drop became not significant at 0.1 nM and 1 nM of Paclitaxel but could still reduce the 20% more of cell at 0.01 µM and 0.1 µM of Paclitaxel upon cotreatment when compare with singlely treated. Colchicine was the most successful western drug to have a significant decrease in the cell viability in cotreatment with S-Rh2. At all concentration of Colchicine tested, there were 30% more cells death in cotreatment with S-Rh2. At 0.01 µM of Colchicine, a further drop of cell viability to 40% was suspected to have a synergistic effect between the two chemicals. The results calculated from Calcusyn software also showed that the comtreatment of S-Rh2 and 0.01 µM of Colchicine had CI vlaue of 0.827 which belongs to the catergory of slight synergism. Nocodazole had an overall drop of 30% cell viability when compared the cotreatment with single treatment of Nocodazole.







Figure 15. Effect of comparision of single treament of western drugs with the cotreatment of ginsenoside S-Rh2 and six western drugs on cell viability on HK-1 cell by MTT assay. HK-1 were cultured and treated with the S-Rh2(50 µM) and six western drugs (0.1 pM, 1 pM, 0.01 nM, 0.1 nM, 1 nM, 0.01 µM, 0.1 µM) at the same time for 48 hours. The extent of cell death was determined and expressed as a percentage of that with untreated control. Data are means ± SD of four independent experiments.


Concentration of conventional drugs

0.1 pM







Docetaxelcotreated with S-Rh2(50µM)








Paclitaxelcotreatedwith S-Rh2(50µM)








Colchicinecotreated with S-Rh2(50µM)








Nocodazolecotreated with S-Rh2(50µM)








Cisplatincotreated with S-Rh2(50µM)








Vinblastinecotreated with S-Rh2(50µM)








Table 1. Effects on HK-1 cancer cell growth of the combination of ginsenoside S-Rh2 and conventional drugs. CI vlaues were calculated according to the Chou and Talalay mathematical model for drug interactions (Chou and Talalay, 1984) using the Calcusyn software. As a measure of interaction, CI values are indicated for different combinations of cotreatment by using MTT assay. CI is a quantitative measure of the degree of interaction between different drugs. When CI value smaller than 0.3, strong synergism. If CI vlaue lines beween 0.3 to 0.7, it represents synergism; CI value of 0.7 to 0.9 indicates slight synergism. If CI vlaues lines between 0.9 to 1.1, it means nearly additive. If CI vlaue is greater than 1, anatagonism.

3.4 Flow cytometric analysis of cotreatment of Rh2 and Colchicine on HK-1 cells

After cotreating S-Rh2 and six conventional drugs, flow cytometry was used to further study the effect of cotreatment of 50 µM Rh2 and 0.01 µM Colchicine on HK-1 cell cycle (Kim et al., 2010) so as to investigate if Rh2 can improve the ability of cell apotosis of Colchicine. Colchicine was found to induce 19.2% of HK-1 cell to ungergo sub-G1 phase apotosis. However, upon cotreatment of 50 µM of S-Rh2 and 50 µM of R-Rh2, the proportion of cells greatly reduced to 8.9% and 10.9% respectively. But the cell percentage in G2 phase was notable. There were 62.5% of HK-1 cells that treated with 0.01 µM Colchicine were in G2 phase. Upon cotreatment of Colchicine with 50 µM S-Rh2 and 50 µM R-Rh2, the proportion increased to 77% and 78.1% respectively.

Sub G1 phase (%)

G1 phase (%)

G2 phase (%)





Colchicine (0.01 µM)




S-Rh2(50 µM)




Colchicine + S-Rh2




R-Rh2(50 µM)




Colchicine + R-Rh2




Table 2. Percentage of cells treated with different compounds in different phase.

Figure 16. Increasment of G2 phase upon cotreatment of S-Rh2 and R-Rh2 with Colchicine. HK-1 were cultured for 24 hours and treated with different drugs for 48 hours. Flow Cytometry assay was carried out to cell cycle analysis.

4. Discussion

MTT is a simple assay which shows the amount of living cells directly which reflects the cell apotosis or cell arrest ability of the drug treatment by comparing to the control treatment (Twentyman and Luscombe, 1987). As many reseaches have been done on ginsenosides which proved that ginsenosides can cause cell cycle arrest on cancer cell like breast cancer cell MCF-7 (Frances et al., 2011) and lung cancer (Gadgeel, 2011), they are said to have the anti-cancer ability. Ginsenosides were also found to enhance the effect of certain conventional drugs like Docetaxel and Cisplatin on cancers, like prostate cancer (Kim et al., 2010) and colon cancer (Kim et al., 2009). In my study, I would like to examine whether ginsenosides has the cell cycle arrest or cell apototic ability towards nasopharyngeal carcinoma as they did on other cancer cells. Also, I want to see if ginsenosides can enhance the anti-prolferation effect of conventional drugs on nasopharyngeal carcinoma so that the concentration that are currently using in the conventional drugs can be lower.

In the eight ginsenosides that I have added to treat HK-1, most of them (S-Rh2, R-Rh2, R-Rg3, PPD, PD, CK and RP-1) showed negative effects on cell viability, ie. they reduced the number of HK-1 cell either by killing HK-1 or inhibiting HK-1 cell growth. These chemicals act on HK-1 cell to different extent at different concentration. The most notable was PD. Almost no cells can survive under high dosage of PD and it has the smallest IC50 value of 25 μM. Similar trends were obtained from three duplicated tests. S-Rh2 and R-Rh2 are stereoisomer, S-Rh2 can kill over 90% of HK-1 at 80 μM but R-Rh2 did not have the same result towards HK-1 with only 10% cells death. This indicats that the three-dimensional orientation is the key that triggers the reaction of cell apotosis (Yuriev et al., 2011). S-Rg3 is the exceptional ginsenoside that showed positive effect on cell viability when high dose was used, ie, promoted cell growth.

Upon cotreament of S-Rh2, all six western drugs had been further strengthened the ability of cell-killing or cell arrest on HK-1 cell. S-Rh2 was selected because of commertial reason and it could compare the results with its enantiomer, R-Rh2, even though it was not as potent as PD. S-Rh2 at concentration of 50 µM was chosen because it was the IC50 value of S-Rh2 which would be more easier to see if there is any synergistic effect in cotreament with western drugs. From the results, all six cotreatments did have a more adverse effect on HK-1. However, it was not great enough to say that they have synergistic effect (1+1>2) as expected. In the cotreatment of 50 µM of S-Rh2 and 0.01 µM of Colchicine, the effect was comparitively drastic with 40% further drop in cell viability. It was the most significant and was suspected to have a synergistic relationship between them. In order to comfirm if the observation was true, analysis of the degree of interaction between S-Rh2 and western drugs was done by Calcusyn software (Morelli et al., 2005). From the result above, most of the combination of drugs (except the cotreatment of Paclitaxel with S-Rh2 and Colchicine with S-Rh2) had a CI value greater than 0.9, which indicated that those combination of drugs did not have synergistic effect. Though the cotreatment of Paclitaxel (0.01 µM and 0.1 µM) and S-Rh2 had CI values less than 0.9, the CI vlaue of cotreatment of Colchicine (0.01 µM and 0.1 µM) and S-Rh2 were smaller (0.827 and 0.811). So, S-Rh2 only have an additive or antagonistic impact with the five western drugs (Docetaxel, Paclitaxel, Vinblastine, Nocodazole and Cisplatin) and have a slight synergistic effect with Colchicine on HK-1 on cell viability. This result apply on the pharmaceutical use to lower the concentration that are currntly used in the conventional drugs while having greater effect on treating cancer.

From the flow cytometry analysis, the sub-G1 peak of cotreatment with Rh2 became shorter when compare with treating Colchicine alone. The proportion of cell also showed the same result upon cotreatment. This represents that neither S-Rh2 nor R-Rh2 can enhance the apototic effect of Colchicine on HK-1 cells. And the slight synergistic effect between Colchicine and S-Rh2 was not caused by promoting cell apotosis. However, both S-Rh2 and R-Rh2 made the G2 peak much higher upon cotreatment than Colchicine was treated alone. Calculation by MOOD Fit program also showed an increasment of more than 20% in percentage of cell at G2 phase under cotreatment of Rh2 than treated with Colchicine alone. This reflects that Rh2 can promote HK-1 cell arrestment at G2 phase. As a result, Rh2 cannot improve the apototic ability of Colchicine on HK-1 but enhance the G2 arrestment ability of Colchicine on HK-1 cell.

5. Conclusion

To conclude, ginsenosides S-Rh2, R-Rh2, PD, PPD, CK, RP-1 and R-Rg3 were found to possess anti-cancer activity on nasopharyngeal carcinoma as they did on other cancer cells. S-Rg3 was found to promote cell growth which is unexpected. Ginsenoside S-Rh2 is not effective to enhance the strength of five conventional western drugs (Docetaxel, Paclitaxel, Vinblastine, Nocodazole and Cisplatin). But it has a slight synergistic effect towards cotreatment with Colchicine. By flow cytometry, Rh2 cannot enhance cell apototic effect of Colchicine but is able to arrest HK-1 cell at G2 phase.