Ann. Cancer Res. Therap. Vol. 15, No. 2, pp.

54-60, 2007

Synergy Effect of Vitamin K3 on Cytotoxicity of Campthotecin in HCT116 Cells

Nobuhiro Yoshimoto, Yuji Takebayashi, Seichi Takenosita
Laboratory of Molecular Medicine, Department of Surgery II, Fukushima Medical University School of Medicine

Abstract Vitamin K is a fat-soluble vitamin involved in clotting factors of blood coagulation system. Recently, various studies about bone metabolism and anti-cancer effect in vitamin K have been reported. The aim of this study was to examine the synergy effect of vitamin K3 (MK0) on the cytotoxicity of anticancer drugs with different pharmacological actions such as campthotecin (CPT), cisplatinum (CDDP), Taxol, 5-FU and VP-16. The synergism of MK0 with anti-cancer drugs in HCT116 cells was evaluated by MTT assay and by analysis with the method of Chou and Talalay. The combination of CPT with MK0 revealed a highly synergistic effect in HCT116 cells. A partial synergism was observed in the combination of 5-FU or CDDP with MK0. In contrast, the combination of MK0 with Taxol or VP-16 resulted in antagonism. Further, 24-h exposure of HCT116 cells to 10 M of MK0 and 10 nM CPT was associated with an increased proportion of cells in S phase, and this combination of drugs promoted arrest in S and/or G2 + M phase, resulting in the enhancement of cytotoxicity of CPT by MK0. In addition, this synergism was taken place at the early phase after the treatment of both drugs by timelapse microscopic analysis. These findings raised the possibility that the chemotherapy of MK0 with topoisomerase I poison may be useful for the improvement of patients with malignancy in clinic. Keywords: Synergy, vitamin K3, menadione, camptothecin

(Received August 17, 2007; Accepted October 12, 2007)

Introduction Vitamin K is a fat-soluble vitamin isolated from a plant in 1939. It is involved in clotting factors (, , , and protein C, S, Z) of normal blood coagulation system by carboxylating glutamic residue in serum. Vitamin K structurally has napthoquinone skeleton and is classified into vitamin K1 (Phylloquinone), vitamin K2 (Menaquinones), vitamin K3 (Menadione) by its side chain structure (Fig. 1). Vitamin K1 is found in many higher plants as well as algae, with the highest concentrations found in green leaf vegetables. Vitamin K2 is also naturally present, however, it is not produced by plants; rather, it is produced by a vast array of bacteria. Vitamin K3 is not considered as a natural vitamin K, but rather a synthetic analogue that acts as a provitamin1). Recently, a lot of studies such as relations with bone metabolism2) and its anticancer effect in addition to clotting factors of blood coagulation system, have been reported on vitamin K. Side chain of vitamin K2, polyprenyl alcohol induces apoptosis3,4). Vitamin K1 and K2 have the ability to inhibit the growth of carcinoma cell lines, however their efficacy is far less than that of vitamin K35). Therefore,
Correspondence to : Yuji Takebayashi, M.D., PhD, Department of Surgery II, Fukushima Medical University School of Medicine, Fukushima, Hikarigaoka 1, 960-1295, Japan, E-mail: tyuji@fmu.ac.jp, Tel: (+81) 24-547-1263, Fax: (+81) 24-548-3249

most studies on anticancer efficacy focus on vitamin K3. It has been demonstrated that Vitamin K3 has anticancer effects in rodents6-10) and human carcinoma cell lines11-13). Several studies showed that oxidizing properties of vitamin K3 generates free radicals, and they break DNA strands14-16) . Vitamin K3 hyperphosphorylated cyclin dependent kinase and arrested cell cycle at G1/S and S/G27,17), and influenced on the expression of oncogenes c-myc and c-fos7,18). Several reports on the combinational effect(s) of vitamin K3 and anticancer drugs are as follow. Vitamin K3 revealed synergism with 5-FU, bleomycin, cisplatin, and dacarbazine in human oral epidermoid carcinoma (KB) cells. Vitamin K3 in KB cells also demonstrated an additive effect in the combinations with another chemotherapeutic agents (mercaptopurine, cytarabine, hydroxyurea, VP-16, vincristine, doxorubicin, mitoxanthine, mitomycin C, actinomycin D, and thiotepa)12). The synergistic action between vitamin K3 and doxorubicin, 5-FU, or vinblastine was also shown in nasopharyngeal carcinoma cells19). The combination of mitomycin C with menadione led to a 10- to 50-fold reduction in the concentration of mitomycin C required for the direct cytotoxicity20) . Pretreatment with vitamin K3 before doxorubicin or mitomycin increased the cytotoxicity of doxorubicin or mitomycin in MCF-7 breast cancer cells21) . However, they analyzed their data, using isobologram method to assess synergy. Because, its absolute premise that drug

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Fig. 1 Chemical structure of Vitamin K (MK0)

vidual IC50 values. Fractional survival (f) was calculated by MTT assay at 540nm absorbance. Data were subsequently analyzed by the method of Chou and Talalay23). In brief, log[(1/f 1)] was plotted against log(drug dose). From resulting median effect lines, the x-axis intercept (log IC50) and slope m (a measure of sigmoidicity) were calculated for each drug and for the combination by the leastsquares method. These parameters were used to calculate the doses of the individual drugs and the combination required to produce varying levels of cytotoxicity (f = 0.95, 0.90, 0.85, 0.05) according to equation 1: Dosef = DoseIC50 [(1-f)/f]1/m [1]

action must be exclusive one another has been a problem and criticized22). The aim of this study was to examine the synergy effect of vitamin K3 on five anticancer drugs with different pharmacological actions such as campthotecine, cisplatinum, Taxol, 5-FU and VP-16 using the method of Chou and Talalay23). We also explored to analyze the mechanism of the synergy of CPT and vitamin K3 in HCT116 cells. Methods Cell line and chemicals. HCT116 cells (provided by Institute of Development Aging and Cancer, Tohoku University, Sendai, Japan), derived from human colon carcinoma, were cultured in RPMI1640 (Sigma-Aldrich Company, St. Louis, USA) with 5% fetal bovine serum. Anticancer drugs such as 5-fluourasil (5-FU), cisplatinum (CDDP), Taxol, camptothecin (CPT), and etoposide (VP-16) were purchased from Sigma-Aldrich Company, St. Louis, USA. Vitamin K analogue, vitamin K3 (menadione; MK0) was provided by Eisai (Company Ltd. Tokyo, Japan). Cell Survival by MTT Assay. Chemosensitivity in vitro was measured by means of MTT colorimetric assay performed in 96-well plate. To determine the effect of 10 l of MK0 with or without 10 l of anti-cancer drugs such as 5-FU, CDDP, Taxol, CPT and VP-16, HCT116 cells were cultured in 180 l of medium. After 24-h incubation (37C, 5% CO2), 20 l of various concentrations of anti-cancer drugs were added, and the plates were incubated for 4 days. Fifty l of MTT (1 mg/ml in PBS) were added to each well. Plates were incubated for 4-h, and the resulting formazan was dissolved in 100 l of DMSO after aspiration of the culture medium. The plates were shaken mechanically for 5-min and read immediately at 570 nm using a model 550 Micro Plate Reader (Bio-Rad, Richmond, CA). Analysis of Combined Drug Effects. In each experiment, cells were treated with serial dilutions of each drug individually and with a fixed ratio of both drugs simultaneously at doses that typically corresponded to 1/2, 5/8, 3/4, 7/8, 1 and 1.5 times of the indi-

When the two drugs were administered at a fixed ratio, the dose of the combination required to produce fractional survival f could be separated into the component doses (D)1 and (D) 2 of drugs 1 and 2, respectively. For each level of cytotoxicity (f = 0.95, 0.90, 0.85, 0.05), a parameter called the combination index (CI) was then calculated according to equation 2: CI=(D)1/(Df)1+(D)2 /(Df)2+(D)1(D)2 /(Df)1(Df)2, [2] where (D)1 and (D) 2 are the concentrations of the combination required to produce survival f, (Df)1 and (Df) 2 are the concentrations of the individual drugs required to produce f, and =1 or 0 depending on whether the drugs are assumed to be mutually nonexclusive, respectively, in their action. According to this method, synergy is indicated by a CI less than, additively by a CI greater than 1. Flow Cytometry. Samples were prepared for flow cytometry. Briefly, cells were harvested, washed in ice-cold PBS (pH 7.4), fixed in ice-cold 70% ethanol, and treated with RNase (500 units/ml; Sigma Chemical Co., St. Louis, MO) at 37C for 30 min. Cellular DNA was stained with 50 g/ ml of propidium iodide (Sigma Chemical Co.), and cells were stored at 4C before analysis. Cell cycle analyses were performed using a Becton Dickinson fluorescenceactivated cell analyzer. Cells (1 x 104) were analyzed for each point, and quantization of cell cycle distribution was performed using Modfit LT (verity Software House, Topsham, USA). Time-Lapse Microscopic Analysis. HCT116 cells were plated in 35 mm glass-bottom culture dishes (MatTek Corporation, MA). After culturing HCT116 cells for 16-h at 37C in 5% CO2, HCT116 cells were incubated with 10 M of MK0, with 10 nM of CPT or with both 10 M of MK0 and 10 nM of CPT. The cell survival was monitored by time-lapse microscopy (IX81, Olympus, Tokyo, Japan) at every one hour for 24-h.

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Results Determination of IC 50 Doses of MK0, CPT, CDDP, Taxol, 5-FU and VP-16. To evaluate the synergy of MK0 with anti-cancer drugs, each IC50 was obtained by MTT assay. IC50 of each drug in HCT116 cells was as follows; MK0/13.4 M, CPT/45.3 nM, CDDP/ 10.2 M, Taxol/ 45.2nM, 5-FU/ 7.4 M and VP-16/ 17.8 M. Simultaneous Exposure to MK0 and CPT, CDDP, Taxol, 5-FU or VP-16. Fig. 2 revealed CI plots in HCT116 cells, which were simultaneously exposed to MK0 and CPT (Fig. 2A), CDDP (Fig. 2B), Taxol (Fig. 2C), 5-FU (Fig. 2D) or VP-16 (Fig. 2E) at equipotent molar ratios described as above (see Materials and Methods). The curves showed synergy or antagonism of the examined drugs in HCT116 cells. CI was below 1, indicating greater than additive effects or synergism. CI was over 1, indicating antagonism. The effect of synergism of MK0 on each anticancer drug was described as followed.

CPT/ Topoisomerase I poison (Fig. 2A). At the levels of cell kill between 10 and 90%, the CI ratios were less than 1 in HCT116 cells treated with MK0 and CPT (upper panel), and with CPT after 24-h incubation with MK0 (middle panel). These results indicated a highly synergistic effect for this drug combination. However, the CI values in HCT116 cells treated with MK0 after 24-h incubation with CPT (lower panel) resulted in higher CI indices, indicating antagonism. CDDP/platinating agent (Fig. 2B). At the levels of cell kill between 30 and 90%, the CI ratios were less than 1 in HCT116 cells treated with MK0 and CPT (upper panel), indicating synergism. Both treatments of HCT116 cells with CPT after 24-h incubation with MK0 (middle panel) or with MK0 after 24-h incubation with CPT resulted in higher CI indices, indicating antagonism. Taxol/antimicrotubule agent (Fig. 2C). In any combination of MK0 with taxol or with VP-16, all CI were more than 1, indicating antagonism. 5-FU/ antimetabolite (Fig. 2D). At the levels of cell kill between10 and 60%, the CI ratios were less than 1 in HCT116 cells treated with MK0 and 5-FU at the same time (upper panel). In addition, at the level of cell kill

Fig. 2 Interaction between vitamin K (MK0) with CPT (A), CDDP (B), Taxol (C), 5-FU (D) and with VP-16 (E) by Chou and Talalay analysis. Treatment of HCT116 cells with MK0 and each drug (upper panel), each drug with after 24-h incubation with MK0 (middle panel), and each MK0 with after 24-h incubation with each drug (lower panel). CI greater than 1.0 indicating antagonism, CI equal 1.0 indicating additively, and CI less than 1.0 indicating synergy.

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Fig. 3 Effect of MK0 with or without CPT on cell cycle in HCT116 cells. Control (A), HCT116 cells were treated with MK0 (B), CPT (C), MK0 and CPT (D), and a 24-h exposure of HCT116 cells to 10 M of MK0 and 10 nM CPT (E).

between 10 and 50%, CI with 5-FU after 24-h incubation with MK0 (middle panel). These results indicated a partial synergistic effect for this drug combination. However, the CI values in HCT116 cells treated with MK0 after 24-h incubation with 5-FU (lower panel) resulted in higher CI indices, indicating antagonism. VP-16/ topoisomerase II poison (Fig. 2E). In any combination of MK0 with VP-16, all CI were more than 1, indicating antagonism. Effects of MK0 and CPT on Cell Cycle Progression in HCT116 cells (Fig. 3 & 4) The highest synergism of MK0 was among the examined anticancer drugs in HCT116 cells was shown in the combination of MK0 and CPT (Fig. 2). To explore the mechanism of this synergism, cell cycle response of HCT116 cells was examined as a function of each drug

Fig. 4 Percentage of cells in S after treatment of HCT116 cells with MK0, CPT , MK0 and CPT, and a 24-h exposure of HCT116 cells to 10 M of MK0 and 10 nM CPT at the indicated time.

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combination showing the synergism. After 12-h exposure of 10 nM CPT to HCT116 cells, 71% of HCT116 cells accumulated in S by the treatment of 10 nM CPT. After 24-h 10 nM of CPT treatment, HCT116 cells progressed through S and were arrested in G2/M (89% of the cells counted). The cell cycle effects of CPT enhanced by MK0 were also studied. After the treatment of MK0 and CPT, HCT116 cells were remained in S (3-h; 47%, 6-h; 59%, 12-h; 71%, 24-h; 62%). Further, HCT116 cells arrested in S (Fig. 4) and the proportion of sub-G1 increased (Fig. 3), suggesting that HCT116 cells were more killed by the enhancement of S arrest by CPT with MK0, compared with that by CPT without MK0. In contrast, after the treatment of CPT after 24-h incubation with MK0, HCT116 cells progressed through S and were arrested in G2 (63% of the cells counted).

Time-Lapse Microscopic Analysis. To observe the synergism of CPT and MK0 more precisely, time-lapse microscopic analysis was performed. The picture was captured at every one hour for 24-h. Representative pictures at 0, 3, 6, 12 and 24-h after the treatment of both CPT and MK0 in HCT116 cells, were shown in Fig. 5A. The enlarged pictures including the area indicated by an arrow in Fig. 5A was shown in Fig. 5B. The dying cells by the treatment of both CPT and MK0 in HCT116 cells were observed in HCT116 cells. The number of alive cells was counted and cell survival ratio was plotted in a graph (Fig. 5C). Interestingly, the cell survival ratio began to decrease after 10-h treatment by both CPT and MK0 and reached at 30% of cell survival ratio until 17-h treatment of these drugs. In contrast, the survival ratio in the cells treated with MK0 or CPT did not reveal the major difference between at 0 hour through at 24-h after the treatment of each drug.

Fig.5 Time-lapse microscopic analysis. A. Representative pictures at 0, 3, 6, 12 and 24-h after the treatment of both CPT and MK0 in HCT116 cells. B The enlarged pictures including the area indicated by an arrow in Fig. 5A. & C. The cell survival ratio was plotted in a graph at the indicated conditions.

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Discussion We examined the synergy effect of vitamin K3 (MK0) on anticancer drugs with different pharmacological actions such as campthotecine (CPT)/ topoisomerase I poison, cisplatinum (CDDP)/ platinating agent, Taxol/ antimicrotubule agent, 5-FU/ antimetabolite and VP-16/ Topoisomerase II poison, using the method of Chou and Talalay. Herein, we found that the combination of MK and CPT revealed a highly synergism in HCT116 cells. Further, a 24-h exposure of HCT116 cells to 10 M of MK0 and 10 nM CPT was associated with an increased proportion of cells in S phase, and promoted arrest in S and/or G2 + M phase, and resulted in the enhancement of cytotoxicity of CPT by MK0. In addition, this synergism was taken place at the early phase after the treatment of both drugs by time-lapse microscopic analysis. The combinational effects of MK0 and anticancer drugs have been reported from several laboratories. Among them, the synergism of MK0 with 5-FU, bleomycin, CDDP, and dacarbazine12), with doxorubicin, 5-FU and vinblastine19), with mitomycin C20), and with doxorubicin and mitomycin21), has been shown in several cell lines. However, their data were analyzed by isobologram. Since the absolute premise of isobologram is that drug action must be exclusive each other, this method for analyzing the mutual drug interaction contains a problem and a criticism22). Therefore, we analyzed the synergism of MK0 with CPT, CDDP, Taxol, 5-FU and VP-16 using the method of Chou and Talalay23). The definite synergism of MK0 with Taxol or VP-16 was not confirmed in this study. As described in the previous reports12,19), the synergism of MK0 with CDDP or 5-FU was observed in HCT116 cells. However, each synergism was very limited in CDDP and MK0 at the levels of cell kill between 30 and 90%, and MK0 and 5-FU at the levels of cell kill between10 and 60%. Of especial interest was the finding that the combination of MK and CPT revealed a highly synergism in HCT116 cells. To explore the partial mechanism(s) of the synergism in the combination of MK0 with CPT in HCT116 cells, the flowcytmetric analysis was performed using he noncytotoxic concentration (10 M) of MK0. During 24-h treatment of MK0, the percent of cells in each phase of cell cycle did not vary. However, a 24-h exposure of HCT116 cells to 10 M of MK0 and 10 nM CPT was associated with an increased proportion of cells in S phase, promoted arrest in S and/or G2 + M phase, and resulted in the enhancement of cytotoxicity of CPT by MK0. In addition, Goldwassser et al proposed two cytotoxic mechanisms of CPT 27) . In their report 27), consistentiy, flow cytometry analyses showed that KM12 cells were arrested in G2, whereas SW620 cells were irreversibly blocked in S phase. Aphidicolin protection was minimal

in KM12 cells and more pronounced in the more sensitive SW620 cells. SW620 cells exhibited also a greater capacity to break through the G2 checkpoint after DNA damage. Their results suggested that misrepair of damaged replicons and/or alterations in DNA damage checkpoints was critical to defining chemosensitivity to CPTinduced top1-cleavable complexes and that CPT appeared to have two cytotoxic mechanisms, one protectable by aphidicolin, and the other not. With these observations and our present data, non-cytotxic concentration of MK0 with CPT may prolonged cells in S phase and MK0 may enhance the cytotocity of CPT in HCT116 cells. In conclusion, we revealed the synergism of MK0 with CPT, 5-FU and CDDP. Of especial interest in the present study was the finding that the combination of MK0 and CPT revealed a highly synergism in HCT116 cells. We further predict that the combination chemotherapy of MK0 with topoisomerase I poison might be useful for the improvement of patients with malignancy.
Acknowledgement
We thank Ms Yuri Konno for her excellent and skillful edition in this manuscript. We also thank Professor K.Ogawa (Department of Surgery, Tokyo Womens Medical University Medical Center East) for his critical reading our manuscript. In addition, a part of this manuscript was presented in 16th Annals of Cancer Research and Therapy.

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