Cancer Res Treat.  2005 Jun;37(3):183-190.

Synergistic Effect of Ionizing Radiation and beta-lapachone against RKO Human Colon Adenocarcinoma Cells

• Kim EJ
• Ji IM
• Ahn KJ
• Choi EK
• Park HJ
• Lim BU
• Song CW
• Park HJ

Abstract

PURPOSE
To reveal the interaction between beta-Lapachone (beta-lap) and ionizing radiation in causing cell death in RKO human colon adenocarcinoma cells, and to elucidate the potential usefulness of combined beta-lap treatment and radiotherapy for cancer treatment. MATERIALS AND METHODS: The cytotoxicities of various treatments were determined in vitro using clonogenic and apoptotic cell death. The changes in cell cycle distribution were studied using flow cytometry and an in vitro kinase assay. The tumor growth was studied using RKO tumors grown s.c. in the hind leg BALB/c- nuslc nude mice. RESULTS: beta-lap caused clonogenic cell death and rapid apoptosis in RKO cells in vitro, in a dose dependent manner. The repair of sublethal radiation damage was almost completely inhibited when cells were maintained in beta-lap during the interval between the two-dose irradiation. Flow cytometry study demonstrated that beta-lap induced apoptosis, independent of the cell cycle phase, and completely prohibited the induction of radiation- induced G2 arrest in irradiated cells. The prohibition of radiation-induced G2 arrest is unclear, but may be related to the profound suppression of the p53, p21 and cyclin B1-Cdc2 kinase activities observed in cells treated with beta-lap. The combination of beta-lap and radiation markedly enhanced the radiation-induced growth suppression of tumors. CONCLUSION: beta-lap is cytotoxic against RKO cells, both in vitro and in vivo, and also sensitized cells to ionizing radiation by inhibiting sublethal radiation damage repair. beta-lap is potentially useful as a potent anti-cancer chemotherapy drug and potent radiosensitizer against caner cells.

Keyword

beta-Lapachone; Ionizing radiation; NQO1; RKO cells; Radiosensitization

MeSH Terms

Adenocarcinoma*
Animals
Apoptosis
Cell Cycle
Cell Death
Colon*
Cyclins
Drug Therapy
Flow Cytometry
Humans*
Leg
Mice
Mice, Nude
Phosphotransferases
Radiation, Ionizing*
Radiotherapy
Cyclins
Phosphotransferases

Figure

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  Fig. 1 Cytotoxicity of β-lap on RKO tumor cells. Cells were incubated with different concentrations of β-lap at 37℃ for varying lengths of time, rinsed with fresh medium, and cultured for 8~9 days with regular medium. The numbers of colonies formed with more than 50 cells were counted, and the %age survival calculated. Each data point shows an average of 7 experiments, from duplicate cultures±1 S.E.

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  Fig. 2 Survival curves for the RKO tumor cells treated with radiation alone (RT only), or with β-lap treatment. Cells were irradiated, incubated with 5 or 10 µM β-lap for 4 h (RT+β-lap) and rinsed. After culturing for 8-9 days, the numbers of were colonies counted and the %age survival calculated. An averages of 5~7 experiments, from duplicate cultures±1 S.E., are shown.

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  Fig. 3 Effect of β-lap on the repair of sublethal damage (SLD) due to radiation. Cells were irradiated with 6 Gy in a single exposure or with two 3 Gy exposures, at 1 h interval in regular media (3 Gy+3 Gy), or in the presence of 8 µM β-lap, between the 2 radiation dose exposures (3 Gy+3 Gy+β-lap). The survivals of the cells exposed to 8 µM β-lap during the 1 h interval between the two irradiation doses were normalized for the cell death caused by β-lap. The means of 5 experiments, with one S.E., are shown.

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  Fig. 4 Apoptotic DNA degradation in RKO tumors cells treated with β-lap alone, irradiation alone and in combination. For the combined effect, the cells were first irradiated and then incubated with 5 µM β-lap for 4 h. Therefore, the '4 h' represents 4 h after irradiation or 0 h after the 4 h β-lap treatment.

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  Fig. 5 Flow cytometric analyses of the cell cycle distribution after irradiation alone, β-lap treatment alone and in combination. For the combined effect, the cells were first irradiated and then incubated with 5 µM β-lap for 4 h. Therefore, the '4 h' represents 4 h after irradiation or 0 h after the 4 h β-lap treatment. The means of 4 experiments, with one S.E., are shown.

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  Fig. 6 The activities of p53 and p21 (A), and that of cyclin B1/Cdc2 kinase (B), in the cells treated with irradiation lone, β-lap alone and in combination. For the combined effect, the cells were first irradiated and then incubated with 5 µM β-lap for 4 h. Therefore, the '4 h' represents 4 h after irradiation or 0 h after the 4 h β-lap treatment. Representative results from 4 studies are shown.

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  Fig. 7 Growth inhibition of RKO tumors in the thigh of BALB/c-nuslc nude mice after various treatments. There were four groups of tumors: (i) Control tumors, (ii) Tumors were irradiated with 10 Gy in a single dose (10-Gy), (iii) Host animals were i.p. injected with 24 mg/kg β-lap and (iv) Host animals were i.p. injected with 24 mg/kg β-lap and the tumor irradiated 30 min later with 10 Gy. The means of 5~7 tumors, with one S.E., are shown.

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