Li Yu Xiong Daishui Gao Min LiDianhong
(Guangdong Provincial Institute of Traditional Chinese Medicine,Guangzhou 510095)
Hu Xiaoke
(Department of Nuclear Medicine,Sun Yat-sen University of Medical Science,Guangzhou510089)

Abstract Objective:To study the effectof coixenolide (CXL) on irradiation to human nasopharyngeal carcinoma cell lineCNE-2Z.Methods:Microcolony formation assay was used in determining sensitivity of CNE-2Zto γ-ray in vitro.Results:CXL shifted the radiation dose-survival curve to the left,withthe decrease of D₀ and D_q values.Radiation dose was reduced by 7.45%～17.3%at D₃₇ level,the sensitizing enhancement ratios (SER)were 1.11～1.46 and 1.02～1.11at D₀ and D_q levels,respectively,when CNE-2Z was treated with 10^-7 ,10^-6.5 and 10^-6 mol/L of CXL.Conclusion:CXL can increase susceptibility of CNE-2Z toirradiation.
Key words Nasopharyngeal neoplasm;Coixenolide;Radiosensitization

Coixenolide(CXL) is a component extracted fromChinese herbal medicine coix seed,and has the actions of anticancer and immunoregulation^[1] .Wepreviously reported that CXL could inhibit the growth of human nasopharyngeal carcinomacells^[2] 。Clinically,however,irradiation is a primary therapy in treatmentof the tumor. The purpose of this paper is to study the effect of CXL on radiation to thecarcinoma cells.

1 Materials and methods

1.1 Cell line Cell line CNE-2Z (established inGuangdong Medical College) was derived from poorly differentiated squamous cell carcinomaof human nasopharynx.
1.2 Drugs and reagents CXL injection (Zhejiang Kanglaite Pharmaceutical Co., LTD.LotNo.98060092-2),RPMI1640(Gibco),calf serum (provided by the Department of Immunology,SunYat-sen University of Medical Science).
1.3 γ-Ray source ⁶⁰ Co radiotherapy machine (America ATC).
1.4 Methods Microcolony formation assay^[3] was used in determingradiosensitivity of CNE-2Z.
CNE-2Z cells in the phase of logarithm growth in RPMI1640 medium with 15%(v/v) ofcalf serum were planted in 96-well microplate in which each well contains 0.2ml of cellsuspension (200 cells).CNE-2Z forming colonies were observed and counted with invertedmicroscope (Olympus IMF-2) 72h after routine culture in CO₂ incubator. A colonyconsisted of 8 or more cells,with clear-cut contour,powerful dioptric property andunanimous cell appearance in morphology.
The experiment in irradiation was made after CNE-2Z was incubated for 16h.CNE-2Z wasirradiated with a single dose of 1～7 Gy respectively,and then culture was kept on for72h.Survival fraction of CNE-2Z was calculated(=irradiated colony numbers/unirradiatedcolony numbers) based on forming colony.A curve was drawn in logarithm of survivalfraction vs radiation dose.The radiation dose-survival curve of CNE-2Z(without drugtreatment)was regarded as the control.In order to investigate the influence of the drug onradiation dose-survival curve,CNE-2Z was treated with CXL at final concentrations of 10^-7 ,10^-6.5 and10^-6 mol/L,respectively, then irradiated in the same manner.In one cultureplate planted with CNE-2Z,of the four groups (24 wells per group) that treated with asingle dose of γ-ray,three were tests for CXL treatment and one was the control.Simultaneously there was a unirradiated group (24 wells)for umerating survivalfraction.Radiation condition was as follows:The culture plate received γ-ray at roomtemperature.Radiation field was rather larger than plate area,i.e.9×13cm*Depth was 1.4cm.The distance from ⁶⁰ Co source to cell layer was 80cm,to plate surface was78.6cm.Dose rate was 1.0Gy/min.
In addition,some other experiments,with three groups (24wells per group) includinggroup of radiation alone(C),group treated with CXL before radiation (T₁ ) andgroup treated with CXL after radiation (T₂ ),were performed to observe theinfluence of the time and order of the drug addition on radiation. CXL was administered toT₁ at final concentration of 10^-6 mol/L,equal volume of medium to Cand T₂ .After incubation for 16h,all groups were irradiated with a single doseof 3 Gy,then T₂ was treated with the same dose of CXL,C and T₁ withmedium.Culture was continued as the above method following irradiation and the colonyformation of CNE-2Z was observed.
1.5 Data analysis and statistics The parameters related to radiation dose-survivalcurve of CNE-2Z were analyzed with the equation S=1-(1-e^-D/Do) )^N ,whereS is the survival fraction,D,radiation dose,D₀ ,the dose that produces 63% ofdecrease of S in the straight part of the radiation dose-effect curve;N represents theinterception in Y axial where the line is extended(the extrapolation value so called) ande is the basic number of natural logarithm.D_o reflects the cell sensitivity tothe ray.The radiation dose-survival curve in this study showed a exponential curve with a“shoulder” which is indicated by D_q ,the threshold dose,and is related tocell repair from sublethal damage. These parameters were calculated according to themethods reported in leteratures^[4～5] .The sensitizing enhancement ratio(SER) equals to D₀ or D_q value of control group divided separatedlyby D₀ or D_q value of test group. Additionally,dose reduction rate (DRR) of ray was numerated at D₃₇ level.D₃₇ is described as theradiation dose that causes S to reduce from 100% to 37%,that is,D₃₇ =D₀ +D_q ,DRR(%)=[1-(D₃₇ value of test group/D₃₇ value of control group)]×100%.Significant differencewas compared using t or F test.n represented the numbers of experiments performed.Allexperiments were repeated 6 times (n=6).

2 Results

The rate of colony formation of CNE-2Z cellswas(67.10±15.33)%(±s,n=6,similarlyhereinafter).Administration of CXL (10^-7 ,10^-6.5 and 10^-6 mol/L)did not influence the proliferation of these cells.The rates were(65.25±15.43)%,(59.32±12.60)%and(68.33±16.65)%,respectively.Variance analysis showed F=0.65<3.10,(P>0.05) incomparison with the preceding datum (control group).After irradiation,CNE-2Z colonyformation was suppressed (decrease in S),which exhibited positive correlation with thedose.Addition of the same doses (see above)of CXL shifted the radiation dose-survivalcurve of CNE-2Z to the left(Fig.1),with the decrease of D₀ and D_q values (Tab.1),the increase of SER and DRR(Tab.2).
In the combination of ray and CXL,treatment of CNE-2Z with the drug(10^-6 mol/L) before or after irradiation (3Gy) caused(46.37±9.49)% and(52.55±12.68)% ofinhibition,respectively.A comparison between the two groups showedt=0.956<1.372,P>0.2.

Fig.1 Effect of CXL on radiation dose-survivalcurveTab.1 Influence of CXL on the parameters related to radiation dose-survival curve(±s,n=6)

Tab.2 Increased radiosensitivity of CNE-2Z Cellby CXL (±s,n=6)

Radiotherapyis given priority to treatment of nasopharyngeal carcinoma.Although radiation therapeutictechnique on this kind of tumor has been continuously improved for the last fewdecades,the best 5-year survival rate still stays around 30%～50%.Recurrence rate afterradiation also remains as high as 30%～40%^[6] .Recurrence is said to berelated mainly to residual tumor after the first course of treatment. Since thesecarcinoma cells are insensitive or resistant to ray,enhancing sensitivity ofnasopharyngeal carcinoma cells to ray is clinically of great significance.
The combination of irradiation with coix seeds for treatment of advancednasopharyngeal carcinoma has shown satisfactory both short term and long term benefits^[7，8] ,suggesttingthat this Chinese herbal medicine may increase the tumor radiosensitivity.The main activecomponent of coix seed is CXL^[1] .Under conditions of this test,low dose ofCXL without cytotoxicity shifted the radiation dose-survival curve of CNE-2Z to the left,with the reduction of D₀ and Dq values.The lowering D₀ means theincreasing susceptibility of CNE-2Z to radiation.Depression of Dq indicates the loweringof threshold dose of ray that kills the cells.Therefore,10^-7 ～10^-6 mol/Lof CXL lowered radiation dose by 7.41%～17.31%,SER also increased with raise of CXLdose.The mechanism of enhancement of radiosensitivity of CNE-2Z remains to be furtherinvestigated.Clinically,sequalae of radiotherary will significantly decrease whenradiation dose is reduced even by 10%,provided the effectiveness unchanges.It is clearthat radiosensitization not only increases effectiveness of therapy,but also improve thelife guality of patients.In addition,the results were similar when CXL was administeredeither before or after irradiation.Thus the clinical use of CXL or coix seeds is notlimited by radiation time.
(The experiment on irradiation in this paper was performed in the Affiliated TumorHospital of Sun Yat-sen University of Medical Science)

[Executive:Deng deling]

Foundation item: This work was supportedby grants from Guangdong Provincial Natural Science Foundation(No.970839) and GuangdongProvincial Administration of Traditional Chinese Medicine (No.97266)
Biography: Li Yu(1958-),male.Graduated from Guangzhou University ofTraditional Chinese Medicine in 1992 and earned a MD degree.Work in the field of cancerresearch and found that coix seed has the action of increasing radiosensitivity ofhuman nasopharyngeal carcinoma.Chief physician at present.

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(Received date:1999-12-01 )