Human adenoid cystic carcinoma multidrug resistance cell line_2754

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Human adenoid cystic carcinoma multidrug resistance cell line_2754

Human adenoid cystic carcinoma multidrug resistance cell line

     Author: Xiao-Dong Zhou Song Guoxiang Sui Zhi

    Fang Ji-Wu Chang

     Abstract Objective: To establish a human adenoid cystic carcinoma of drug-resistant cell line, in order to clarify the adenoid cystic carcinoma of the reversal of multidrug resistance mechanisms and to provide models and research basis for drug resistance.

     Methods: vincristine (VCR) as the induction agent, by increasing the concentration of intermittent stimulation of

    human adenoid cystic carcinoma cell line (ACC) in vitro induced resistance to the establishment of resistance to VCR of adenoid cystic carcinoma cell line ACC / VCR. Cell count cell growth curve mapping, thiazole blue (MTT) colorimetric

    assay cells to chemotherapeutic drug sensitivity. Results: ACC in vitro after induction, ACC / VCR cells to vincristine (VCR), adriamycin (ADM) and pingyangmycin (PYM) markedly enhanced resistance, with cross-resistance of cyclophosphamide

    ( CTX), fluorouracil (5-FU) and cisplatin (DDP) resistance

    without significant changes. Resistance before and after the ACC cell growth curve, population doubling time and the light microscope, no significant changes in patterns. Conclusion: VCR adenoid cystic cancer cells can induce multi-drug


     Key words adenoid cystic carcinoma multidrug resistance of vincristine

     Establishment of multidrug cell line of human adenoid

cystic carcinoma

     Abstract AIM: To establish an in vitro resistance

    model of adenoid cystic carcinoma which can provide theoretical information for elucidating the mechanisms of multidrug resistance (MDR), improving the chemotherapy scheme, evaluating the prognosis and reserving the MDR in adenoid cystic carcinoma.METHODS: The resistance cell line, ACC / VCR, was induced in the ACC cell line in vitro by progressive concentrations of vincristine (VCR), a drug of choice in the treatment of adenoid cystic carcinoma. The growth curve, drug sensitivity in the parental and resistant cell lines were

    investigated by cell counting and MTT assay. RESULTS: ACC / VCR cell line was cross-resistant to vincristine (VCR),

    adriamycine (ADM) and pingyangmycin (PYM), but not resistant to Cyclophosphamide (CTX), fluorouracil (5 -- FU) and

    cisplatin (DDP). There was no significant difference in the doubling time and morphological changes between ACC cell line and ACC / VCR cell line. CONCLUSION: Exposing to VCR can induce MDR in adenoid cystic carcinoma.

     * KEYWORDS: adenoid cystic carcinoma; multidrug

    resistance; vincristine

     Zhou XD, Song GX, Sui ZF, Chang JW. Establishment of multidrug cell line of human adenoid cystic carcinoma. Int J Ophthalmol (Guoji Yanke Zazhi), 2007; 7 (2) :417-419

     0 Introduction

     Adenoid cystic carcinoma (adenoid cystic carcinoma, ACC) is a highly malignant epithelial tumors, can occur in various parts of the body, such as the salivary gland, submandibular gland, trachea, lung, breast, skin, and so on, in the eyes then occur in the lacrimal gland [1]. The current treatment of adenoid cystic carcinoma in an integrated treatment of the main, in which chemotherapy primarily as a neo-adjuvant

    chemotherapy (pre-operative chemotherapy or radiotherapy

    before surgery), adjuvant chemotherapy (chemotherapy after

    surgery or radiotherapy), radiotherapy sensitizers and palliative chemotherapy applications [2]. Some tumor cells of a chemotherapy drug resistance in the same time, other

    structures and mechanisms of different chemotherapy drugs also produced resistance, known as the tumor cells of multi-drug

    resistance (multidrug resistance, MDR) [3]. The emergence of multi-drug resistance leading to tumor drug resistance and chemotherapy, one of the important reasons of failure. Through the establishment of adenoid cystic carcinoma induced by

    vincristine resistant cell line, is designed to clarify the adenoid cystic carcinoma of the multi-drug resistance

    mechanisms to guide multi-drug resistance reversal agents of

    the research models and research data.

     1 Materials and methods

     1.1 The materials were adenoid cystic carcinoma (ACC) cell lines can be purchased from Shanghai Shen gaming companies. RPMI1640 powder medium (Gibco Company), fetal calf serum (Tianjin Hao Yang Bio Co., Ltd., 56 ? 40min inactivated

    complement), blue, streptomycin (Shijiazhuang Pharmaceutical Factory), glutamine (L-glutamina) ( Gibco Company), trypsin

    (Sigma Corporation), EDTA (EDTA) (Guangzhou Chemical Reagent Factory), dimethyl sulfoxide (DMSO) (Beijing Ding States Biological Co., Ltd.), methyl thiazolyl tetrazolium (MTT) (Beijing Ding States Bio Co., Ltd.), vincristine (vincristine, VCR) (Shanghai Hualian Pharmaceutical Co., Ltd.), cyclophosphamide (CTX) (Shanghai Hualian Pharmaceutical Co., Ltd.), doxorubicin (adriamycin, ADM) (Shenzhen Wan Yue

    Pharmaceutical Co., Ltd.), fluorouracil (5-FU) (Tianjin

    People's pharmaceutical companies amino acids), BLM (PYM) (Tianjin Taihe Pharmaceutical Co., Ltd.), cisplatin (DDP) (Shandong Qilu Pharmaceutical Factory). CO2 incubator (Heraeus, Germany), inverted microscope (Olympus-1M, Japan),

    bio-microscope (Olympus BH-2, Japan), UV spectrophotometer

    (UV754, Shanghai Analysis Instrument Factory), desktop, high-

    speed centrifuge ( Allegra64R, the U.S. Beckman), magnetic stirrer (Jintan City, Jiangsu Province, Tongji Instrument

    Factory), enzyme-linked instrument (DG-3022A, Nanjing tube


     1.2 Methods ACC cell lines cultured in containing 100ml / L inactivated fetal calf serum, 100kU / L blue, streptomycin RPMI1640 culture medium. Culture conditions: 37 ?, 50ml / L

    CO2, every 3d for fluid 1, 2.5g / L trypsin, 0.2g / L EDTA digestion and passage, about 5d passage 1. Intermittent stimulation with increasing concentrations of VCR-resistant

cell selection, concrete steps are as follows: VCR 15μg / L

    × 7d, to drug 7d, VCR 30μg / L × 7d, to drug 7d, VCR 60μg

    / L × 7d, to drug 7d, VCR 60μg / L × 7d, to drug 7d, VCR

    120μg / L × 7d, to drug 7d, VCR 120μg / L × 7d, to drug

    7d, VCR 250μg / L × 7d, to drug 7d, VCR 250μg / L × 7d, to

    drug 7d, drug-free after 7d culture experiments. The growth of cells to take in good condition, 2.5g / L trypsin, 0.2g / L EDTA digestion and made cell suspension, counting, the cell suspension was inoculated into 24-well plates, each hole 1mL,

    inoculation concentration of 1 × 107 / L, 37 ?, 50ml / L CO2

    culture. Cells per day to take three plate counts to calculate the mean. Continuous observation of 7d, during training 3d cell count after the change to non-liquid. With culture time

    as the horizontal axis, cell number of the vertical axis

    (right number), in the semi-logarithmic coordinate paper, draw

    the growth curve, according to formula cells in the logarithmic growth phase the doubling time. TD (doubling time) = t (culture time, h) × lg2 / (lgNt-lgN0). N0, Nt: after

    vaccination and cultured cell number after t h. MTT colorimetric test [4] Determination of sensitivity to chemotherapeutic drugs: logarithmic growth phase cells, 2.5g / L trypsin, 0.2g/LEDTA digestion, containing 100ml / L fetal calf serum in RPMI1640 medium dubbed in 5 × 106 ~ 1 × 107 /

    L in single cell suspension; take 96-well plate, each hole by

    adding 200μL cell suspension, 37 ?, 50 ml / L CO2 cultured

    overnight; replacing culture medium, according to a series of concentrations (final concentration of To 0.01,0.1,1,10,100,1

    000 mg / L) to join anticancer drugs, each repeated three hole concentration in order to not only increase cell culture medium of the hole as a blank control, 37 ?, 50 ml / L CO2

    cultured 72h; each conga 5g / L MTT solution, 20μL, 37 ?, 50

    ml / L CO2 were incubated for 4h, the termination of culture, be careful suction hole supernatant discarded, each hole by adding dimethyl sulfoxide (DMSO) 150μL, oscillation 10min, to

    make it fully dissolved; choice 490nm wavelength, the hole was measured by enzyme-linked instrument on the absorbance (A); according to a formula cell survival to cell survival rate of drug concentration on the number of mapping, find the half inhibitory concentration of drug (IC50), calculated resistance factor (resistance factor, RF). Cell survival rate (%) = absorbance value of test group A / control absorbance value A × 100%, RF = resistance cell IC50 / parental cells IC50.

     2 Results

     2.1 cell line ACC cell lines in vitro induced by VCR screening, continuous culture 4mo, obtained a 250μg / L VCR

    grow well under the action of the drug-resistant cell lines.

    Named the ACC / VCR. No drugs culture 2mo, resistance index of the VCR was no significant decrease, indicating a steady resistance. VCR-induced drug resistance in vitro cultivation

    of the process, dosing of cell swelling, cytoplasmic granules coarse, inhibition of cell growth and proliferation of very slow or even non-proliferation, followed a large number of off wall, decrease in the number. Change with the cultivation of

    hydroponic culture without VCR, and an appropriate extension of passage cycles, surviving the growth of drug-resistant cell

    division accelerated the formation of multiple cell clones, and then gradually integrated into the film, but the cell

    sizes arranged in disorder and atypia is more obvious. And then continue to use the VCR-containing culture medium

    screening. After several screening, non-resistance or low

    resistance of the cells are excluded, the remaining is to have a higher resistance cells.

     2.2 The cell morphology ACC and the ACC / VCR cells were adherent in vitro growth, in the inverted microscope for the polygonal epithelioid cells, the size is more consistent, within the cytoplasm shows the depth of penetration, ranging from light particles, nucleus, class round or irregular shape, nuclear / high quality ratio, nucleolus 2 ~ 3, clearly visible, and seen a huge fusion cells. Cell division relative to more common, and multiple-level nuclear fission, when the

    tightly packed cells embedded into one another like paving stones, the density increased again when overlapping growth, manifested as disappearance of contact inhibition (Figure 1A, B). ACC and the ACC / VCR cells in vitro proliferation rate similar to (growth curve shown in Figure 2). Population

    doubling time was 28.32 ? 1.65h and 27.55 ? 0.89h, between

    the two groups showed no significant difference (t = 1.162, P = 0.311).

     2.3 The resistance spectrum of ACC / VCR not only to the original with a VCR-induced drug resistance, but also to ADM,

    PYM has cross-resistance (Table 1). The amount of various chemotherapeutic drugs commonly used by clinical blood concentrations were reached: VCR 0.25mg / L; CTX 140mg / L; ADM 6mg / L; 5-FU 50mg / L; PYM 20mg / L; DDP 5mg / L.

     3 Discussion

     In recent years, studies have found that tumor cells were more commonly on the natural plant base and its semisynthetic derivatives (such as vincristine, vinblastine, colchicine, etc., etoposide, etc.) and antibiotics (such as A sp Su,

    actinomycin D, daunorubicin, light God ADM, etc.) is easy to produce drug resistance and cross-resistance, and different

    tissue-derived tumors occurring multi-drug resistance

    mechanism is also different [5] . Pairs of MDR research for improving the prognosis of cancer patients to develop a

    reasonable treatment options in order to prevent the emergence or MDR reversal MDR of great significance. The establishment of drug-resistant cell lines in vitro were seen at home and abroad is reported that commonly used method is increasing the

    concentration of intermittent stimulation [6,7], namely, by in vitro cultured parental cells gradually increased the concentration of a certain kind of chemotherapy drugs, through the role of drug choice access to drug-resistant cell line, in

    order to provide in vitro model of multi-drug resistance. Of

    course, the drug in vivo than in vitro metabolism and the role of the complexity of the process and should not simply ignore the experiments in vitro studies of clinical samples. Reposted

    elsewhere in the paper for free download http://

     The human ACC cells in vitro studies have reported that more than at home and abroad, has been to establish a SACC83, ACC-M and other cell lines, and carried out a series of drug susceptibility testing, radiation therapy, biological therapy in experimental study [ 8,9]. However, in vitro environment, the body is far less complex, and in vitro process, certain biological characteristics of tumor cells may change with the actual process may have some access, so the conclusions in

    vitro experiments can not fully explain the sometimes tumors in the body of the biological behavior and pathological processes. As tumor cells, ACC unlimited cell lines passaged in vitro can be unlimited growth, but lose in the course of

    the original culture of the tubular-like arrangement of

    structures, paved sheet, close to the solid-type cell array

    structure. We vincristine as induction agent, intermittent stimulation by increasing the concentration of ACC cell lines

    in vitro screening and induction of resistance, set up VCR-

    resistant adenoid cystic carcinoma cell line ACC / VCR. VCR is

    the ACC most commonly used chemotherapy drugs, is one of the most vulnerable to chemotherapy drugs produce a kind of resistance. At home and abroad with the establishment of multi-drug-resistant tumor model reports, VCR is one of the most commonly used drugs, increasing the concentration of intermittent stimulation is the most common induction method, which with the clinical use of chemotherapy drugs have a

    certain resemblance [ 6]. Through the establishment of drug resistance in vitro model can multi-drug resistance mechanisms

    of cancer research, used to explain the cancer mechanism of drug resistance occurring in the body, but also that

    resistance reversal agents provide the basis for the study. ACC and the ACC / VCR cells in the growth curve and population doubling time were no significant changes in the inverted microscope, no significant change in cell morphology. However, some studies show that the electron microscope parental cells and drug-resistant cells are morphological changes. Prados et al [10] In the study, actinomycin D (DAC) established for the inducing agent resistant rhabdomyosarcoma cell line was found by electron microscopy the cytoplasm of drug-resistant cells

    have aggregation component filaments, organelles increased, double-leaf nuclear and other changes, the cell morphological changes are mainly reflected in the ultrastructural.

     Drug Susceptibility of malignant tumor treatment has been

    the study of the hot issues, accurate and rapid tumor chemotherapy drug sensitivity test to guide selection of clinical chemotherapy drugs, improve the efficacy of great significance [11,12]. But mainly in laboratory studies, the

    real entered clinical prospective study to guide clinical treatment is still limited, especially in China, it has yet to conduct clinical trials reported. At present, for the clinical study of effect of chemotherapy in ACC are divided, for the choice of chemotherapy vary greatly. This may be associated with tumor biological characteristics (histological type, differentiation and cell kinetics, etc.), the patient's individual differences (for chemotherapy sensitivity and tolerance levels), the drug itself and other related toxic

    reactions. Are often used in clinical chemotherapy in the treatment of the same kind of tumors with different patients and even patients suffering from various tumors, which is obviously not scientific, for different tumors in patients

    with drug sensitivity test to select effective chemotherapy drugs, to develop individualized chemotherapy regimens,

    chemotherapy is the future trend of development. We selected 6 kinds of chemotherapy drugs, different mechanism of action, and are now commonly used clinically for the treatment of ACC of the chemotherapy drugs [13]. Since 1983, Mosmann the first study of the use of MTT has been the living conditions of the tumor cells, MTT method and the rapid establishment of drug sensitivity test be developed [4]. The dye is a hydrogen atom can accept dyes, chemical name 3 - (4,5 - dimethyl-thiazol -2)

    -2,5 - diphenyl tetrazolium salts (3 - (4,5 --

    dimethylthiazol-2) -2,5-diphenyltetrazo-diumbromide, MTT),

    trade name thiazole blue, referred to as MTT. MTT method is

    characterized by high sensitivity, fast, simple, economic, without radioisotopes, the results of good reproducibility and can be also multi-drug screening and observation, applicable to all tumor types, the current U.S. National Cancer Institute

    which have been the conventional chemotherapeutic drugs as a screening tool. We use MTT method in vitro drug sensitivity test, results showed that parental cells induced by the drug-

    resistant cells derived ACC / VCR resistant not only to the VCR, but also to ADM, PYM have taken place in cross-

    resistance. This study suggests that multi-drug resistance is

    currently prevalent in the natural sources consistent with the views of chemotherapy drugs. By MTT test we found that chemotherapy drugs reached routine clinical use of blood

    concentration of the majority of the tumor cells fail to effectively inhibited, but through the unlimited increase in drug use in order to achieve the purpose of the practice of killing tumor cells can only be used for in vitro experiment,

    in clinical medicine blindly increasing the amount of chemotherapy, will greatly increase the body's toxic side effects may even lead to fatal consequences. There are two solutions, one through the reversal of resistance to pharmaceutical research and development, to increase tumor

    cell sensitivity to chemotherapeutic drugs in order to reduce the dosage to alleviate the toxic side-effects; second is

    research and development of new drug dosage form, with the targeting of drug and slow release characteristics of tumor tissue can be achieved partially effective blood drug concentration in peripheral blood drug concentration is very low. For example, the development of a new multi-drug

    resistance reversal agents for multidrug resistance-related

    genes are known to reverse drug resistance gene therapy [14], as well as the effect of using a sustained-release

    chemotherapy drugs wrapped in nanoparticles targeted therapy

of active [15], will be the new hot spot for future



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