Empirical Study on Chemotherapeutic Susceptibility of SP Cells in Human Pulmonary Adenocarcinoma Cell Line A549 in Vitro

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1 Clin Oncol Cancer Res (1) 7: DOI 1.17/s Empirical Study on Chemotherapeutic Susceptibility of SP Cells in Human Pulmonary Adenocarcinoma Cell Line A549 in Vitro Tong XIE Li LI De-seng LIU Nai-quan MAO Dan-rong LI Chuan-tian ZUO Ding-ming HUANG Department of Thoracic Surgery, the Affi liated Cancer Hospital of Guangxi Medical University, Nanning 5321, Guangxi Province, China. Correspondence to: Tong XIE Tel: OBJECTIVE To explore the chemotherapeutic susceptibility of SP cells in human pulmonary adenocarcinoma cell line A549 and the possible mechanism of multidrug resistance. METHODS SP and non-sp () cells in the cell line A549 were isolated by fluorescence activated cell sorter. The susceptibility of SP and cells to DDP, 5-FU, VP16, NVB and GEM was detected by a drug susceptibility test, and IC 5 s were calculated 24 h a er the chemotherapy; and then a er a 2-hour IC 5 treatment with 5 chemotherapeutic drugs on the 2 subsets of cells, the intracellular drug levels were determined and analyzed using high performance liquid chromatograph. RESULTS There was no statistical significance in comparison of the differences in IC 5 s and in intracellular drug levels a er DDP treatment between the 2 subsets (P >.5), (P >.5). However, all IC 5 s of the other 4 drugs were significantly higher in the SP cells than in the cells (P <.1). A er the chemotherapy, the intracellular drug levels of the other 4 drugs were significantly lower in SP cells than in cells (P <.1). CONCLUSION Compared to cells, SP cells in the cell line A549 have stronger resistance to the chemotherapeutics. The multidrug resistance of SP cells closely correlates with the function of SP cells discharging chemotherapeutic agents. KEY WORDS: neoplastic stem cells, abcg2 protein, human, drug resistance, multiple, intracellular space, drug tolerance. Copyright 1 by Tianjin Medical University Cancer Institute & Hospital and Springer Introduction Received May 3, 1; accepted August 1; cocr@gmail.com Tel (Fax): Multidrug resistance (MDR) is a big barrier affec ting the curative effect of malignant tumors. It has been shown more and more in researches that the tumor stem cell (TSC) is the starting cell of tumorigenesis [1-3], and may possibly be the major reason for tumor cells developing drug resistance [4]. It can, therefore, be predicted that overcoming the drug resistance of TSC will be of far-reaching significance in cancer treatmnet. Quite a few findings demonstrated that there is an accumulation of TSC in the SP cell subset of the malignant tumors [2,5,6]. Therefore, the SP cell subset in the human pulmonary adenocarcinoma cell line A549 was separated out, and in vitro susceptibility test and intracellular fluid retention monitoring were conducted based on the confirmed characteristics of the stem cells and over-expression of ABCG2 [7], in order to investigate the

2 266 Clin Oncol Cancer Res (1) 7: impact of the SP cell subset on the resistance of cell line A549 and potential drug resistant mechanism. Materials and Methods Main materials and reagents The human pulmonary adenocarcinoma cell line A549 was purchased from the Cell Bank of Peking Union Medical College, China; fetal calf serum (FCS) from Tianjin Haoyang Biological Product Co., Ltd.; DMEM from Gibco/BRL, USA; Hoechest33342, verapamil and propidium iodide (PI) from Sigma Co., Ltd. USA; CCK-8 kit from the Beyongtime Bio Tech Inst., Haimen, Jiangsu, China; cisplatin (DDP) ; 5-fluorouracil (5-FU); etoposide (VP16); vinorelbine (NVB) and gemcitabine (Gem). Cell culture The cells A549 were cultured in the DMEN culture fluid containing 1% FCS, 1 U/mL penicilin and 1 U/ ml streptomycin at 37 C with saturated humidity of 5% CO 2. Digestion and passage for a 1:1 mixed liquor of.2% EDTA and.25% trypsin were conducted. Separation of the SP and non-sp subsets in the cell line A549 FACS Ventage flow cytometry was used to sort out the SP and cell subsets in the cell line A549. The A549 cells at logarithmic phase were collected to prepare the mono-cellular suspension through centrifugation followed by washing twice with PBS. Resuspension was conducted using the DMEN culture fluid containing 2% FCS, and cell account was adjusted to /ml. In one group, Hoechst was added until the final concentration reached 5 mg/l. In the other group, verapamil was added together with Hoechst up to the final concentration of 75 mg/l, followed by water bath at 37 C for 9 min, then discontinuous shaking for uniformity. After 5-min centrifugation at 1 r/min, the supernatant was discarded. The cells were washed once using the PBS containing 2% calf serum, and were re-suspended in the ice-cold PBS containing 2% calf serum. After adding PI up to the final concentration at 2 mg/l, the cells were detected and sorted out using flow cytometry. The excitation light of Hoechst was 352-nm ultraviolet light. The blue light was collected using 424/44 band pass and the red light was collected by 63/22 band pass. The excitation light of PI was 488-nm blue light and the red light was collected using 575/26 band pass. Susceptibility test of the drugs against the 2 subset cells (CCK-8 method) Susceptibility test of the drugs and measurement of intracellular drug levels in the 2 subset cells: i) susceptibility test: the 2 subset cells were harvested respectively and inoculated into a 96-well plate based on cells per well. After the cells were cultured for 12 h, the chemotherapeutic agents were added. In each drug, there were 5 dilutions of different strengths, with 3 parallel wells for each strength dilution, and the blank and control wells. After another 24 h of cell culture, CCK-8 solution was added and incubated for 1 h. The Bio- Rad enzyme-labeled immunosorbent assay was used to determine the absorption values in the wells, and a colorimetric assay with a 45-nm wavelength of laser was chosen to compute the inhibition ratio of the drugs in different concentrations. The analytic graph was drawn up with the X-axis representing the drug level and Y-axis signifying the inhibition ratio; ii) measurement of intracellular drug levels in the 2 subset cells: after harvested, the 2 subset cells were inoculated into a 6-well plate at per well and then cultured for 12 h. Based on the result of the susceptibility test, DDP (1 μg/ml), 5-FU (1 μg/ml), VP16 (1 μg/ml), NVB (7 μg/ml) and GEM (7 μg/ml) were respectively added to the solution and cultured. Administration of the drugs was discontinued 2 h after culturing. The eluant was discarded after each of 3 times PBS washing in the groups. Then.5 ml distilled water was added, and repeated freeze thawing at - C was conducted, in order to rupture tumor cells, until no intact cells were found under microscope. Centrifugation was conducted at 1 rpm for 5 min, and after the supernantant was taken out, the high performance liquid chromatogram detection was performed. Statistical analysis SPSS13. was used for statistical data processing. IC 5 was calculated using the Probit probability ratio regression. The t-test was used for comparing the IC 5 and intra-cellular drug levels between the 2 subsets. Results Sensitivity test for chemotherapeutics in treating the 2 subsets For the sensitivity of the 2 subsets to DDP, please see Fig.1. IC 5 of SP cells = ± 9.53 μg/ml, IC 5 of cells = ± μg/ml. There were statistically significant differences between the 2 groups (P >.5). Drug-resistant test of and cells on DDP Fig.1. Result of drug sensitivity test for the 2 subsets to DDP.

3 Clin Oncol Cancer Res (1) 7: For the sensitivity of the 2 subsets to 5-FU, see Fig. 2. IC 5 of SP cells = ± μg/ml, IC 5 of cells = ± 7.11 μg/ml. There were statistically significant differences between the 2 groups (P <.1). For the sensitivity of the 2 subsets to NVB, see Fig.4. IC 5 of SP cells = 92.9 ± 2.61 μg/ml, IC 5 of cells = 69.4 ± 1.89 μg/ml. There were statistically significant differences between the 2 groups (P <.1). Drug-resistant test of and cells on 5-FU Fig.2. Result of the drug sensitivity test for the 2 subsets to 5-FU. For thedrug sensitivity of the 2 two subsets to VP16, see Fig.3. IC 5 of SP cells = ± 3.2 μg/ml and IC 5 of cells = ± 9.34 μg/ml, There were statistically significant differences between the 2 groups (P <.1). Drug-resistant test of and cells on VP Fig.3. Result of drug sensitivity test for the 2 subsets to VP16. Drug-resistant test of and cells on GEM Fig.4. Result of test in drug sensitivity test forof the 2two subsets to NVB. For the sensitivity of the 2 subsets to Gem, see Fig.5. IC 5 of SP cells = ± 4.21 μg/ml, IC 5 of cells = 75.3 ± 2.8 μg/ml. There were statistically significant differences between the 2 groups (P <.1). Drug-resistant test of and cells on NVB Fig.5. Result of drug sensitivity test for the 2 subsets to GEM. a b c Fig.6a. DDP standard substance Fig.6b. DDP level in SP cells Fig.6c. DDP level in level cells a b c Fig. 7a Level of 5-FU standard Fig. 7b 5-FU level in SP cells Fig. 7c 5-FU level in substance cells

4 268 Clin Oncol Cancer Res (1) 7: a b c Fig. 8a Level of VP16 standard Fig. 8b VP16 level in SP cells Fig. 8c VP16 level in substance cells a b c Fig.9a Level of NVB standard. Fig. 9b NVB level in SP cells. Fig. 9c NVB level in cells. a b c Fig.1a Level of GEM standard. Fig.1b GEM level in SP cells. Fig.1d GEM level in substance cells. Measurement of intracellular drug levels Standard curves showed that the DDP level was.9 ±.7 μg/ml in the SP cells and was.947±.47 μg/ml in the cells. There were no statistically significant differences between the groups (P >.5), see Fig. 6a-c. The 5-FU level was.537 ±.51 μg/ml in the SP cells, and was.98 ±.46 μg/ml in the cells. There were no statistically significant differences between the 2 groups (P <.1), see Fig.7a-c. The VP-16 level was.491 ±.8 μg/ml in SP cells and was.787±.16 μg/ml in cells. There was statistically significant differences between the 2 groups (P = <.1), see Fig. 8a-c. The NVB level was.986 ±.56 μg/ml in SP cells, and was 2.4 ±.718 μg/ml in cells. There was statistically significant differences between the 2 groups (P <.1). See Fig.9a-c. GEM level was.179 ±.9 μg/ml SP cells, and was.371 ±.13 μg/ml in cells, with statistically significant differences between the 2 groups (P = <.1). See Fig.1a-c. Discussion SP cells are a kind of cells that can remove the fluorescent reactive dye Hoechst out of the cells, and were first discovered by Goodell et al. [8] in their study on the cell cycle. They found that the SP cell in the murine bone marrow had the characteristics of the hematopoietic stem cell. It has been shown in the present studies that the characteristic of the SP cell s discharging Hoechst depends on the MDR gene ABCG2/ BCRP [9]. There are quite a few findings on the MDR of antagonizing chemotherapeutics in the TSC, with the key basis mainly being from 3 aspects. Firstly, there is a close relationship between the super-family of the ABC transport protein and TSC. It has been confirmed in

5 Clin Oncol Cancer Res (1) 7: previous studies that there is an over-expression of TSC or super-family of the ABC transport protein in SP cells enriching stem cell, and the over-expression of the ABC transport protein in these SP cells presents the MDR phenotype through the effects of the chemotherapeutics. For example, the SP cells isolated from the liver cancer cell line HuH7 present a high expression of ABCG2 and ABCB1, resulting in the SP cells developing resistance to adriamycin, 5-FU and Gem [1]. Also, in the SP cells isolated from the ovarian cancer cell line Movcar 7, the phenotype of resistance to adriamycin was presented because of the over-expression of BCRP-1 [11]. In the lung cancer cell SN-38, there is a converse relationship between the concentration of topotecan and expression level of BCRP-mRNA, suggesting that the expression of BCRP-mRNA reflects the activity of BCRP protein [12]. Secondly, some growth factors and anti-apoptotic signaling molecules expressed or activated by the stem cells may also cause an increase of MDR in the stem cell [13]. Thirdly, it has been also found in the in vitro test for the drug sensitivity that there is a stronger tolerance to cytotoxic drugs in the SP cells than in the cells. Hirshmann-Jax et al. [14] pointed out that only part of the cells had the SP phenotype and had the expression of the ABCG2, and the drug sensitivity test for the commonly used anticancer drugs was stronger in cells than in SP cells of the tumor, showing that the SP phenotype of the tumor cells was the direct evidence which results in a decreased drug sensitivity. It has been confirmed by the results of the present studies that the sensitivity of the SP cell subset to 5-FU, VP16, NVB and Gem is significantly lower than that of cell subset, and that intracellular drug level detection also reveals lower drug level of the 4 chemotherapeutic agents in the SP subset cells than in the subset cells, indicating that ABCG2 plays an important role in stimulating the SP subsets to discharge the abovementioned chemotherapeutic agents from the cells. It is the discharging of the drugs that directly results in the SP cells developing tolerance to the chemotherapeutic agents. Especially, the results of intracellular drug level tests provide direct experimental evidence for the theory that the tolerance is one of the characteristics of TSC. Dean et al. [4] put forward 3 drug-resistance modes of TSC as follows: i) the stem cell itself is drug resistant because TSC is usually at the stationary phase; has strong ability to repair DNA and can express the superfamily of ABC transport proteins. Part of TSCs may survive after chemotherapy, and then proliferate to develop recurrent tumors; ii) in the acquired drug-resistance of the stem cell, based on the same mechanism of mutation-accumulation in normal stem cells, new drug resistance occurs in the stem cells and the adjacent daughter cells by gene mutation, gene activation and gene amplification etc., after a long exposure of TSC to irradiation and one of carcinogens. iii) collectively inborn resistance: the inborn tolerance exists in TSC and all kinds of differentiated cells, therefore, there is no or very little drug effects in all tumor cells. Our results demonstrated that the MDR mechanism of SP cell subset in the human pulmonary adenocarcinoma A549 cells is in accordance with the first mode mentioned above by Dean et al. Conversely, it is worth noting that the sensitivity of SP cell subset to DDP is quite similar to that of cell subset. There are no statistical differences in the IC5s of drugs to DDP between the 2 subsets. The measurement of intracellular drug levels also indicates that there are statistically significant differences in DDP levels between the 2 subsets. Honjo et al. [15] found in their study on the drug-fast cell line with over-expression of the ABCG2/BCRP gene resulting from Mitoxantrone induction that the gene mutation of ABCG2/BCRP could change the characteristics of the induced MDR. Based on Honjo s finding, we can infer that the binding site of DDP may be lacking in the wild-type ABCG2, resulting in the failure of exerting the function on discharging DDP. Therefore, the SP and cells are both sensitive to DDP. This can partially explain why therapeutic alliance with cisplatin drugs is usually used as the first-line treatment in chemotherapeutic regimens for lung cancer. Thus, clinical test for ABCG2 before starting chemotherapy can be the guideline for planning an individualized chemotherapy regimen for lung cancer patients, and for predicting the chemotherapeutic efficacy and prognosis. The results of the study also reflect that ABCG2 is probably effective in treating lung cancer. Further research, development and use of the high performance ABCG2 inhibitors or reversal agents would help to improve the treatment effects of lung cancer. Conflict of interest statement No potential conflicts of interest were disclosed. References 1 Al-Hajj M, Wicha MS, Benito-HemandezA, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci 3; 1: Kondo T, Setoguchi T, Taga T. 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