F-01A, an antibiotic, inhibits lung cancer cells proliferation

Chinese Journal of Natural Medicines 2014, 12(4): 0284−0289

Chinese Journal of Natural Medicines

F-01A, an antibiotic, inhibits lung cancer cells proliferation WANG Jing 1, 2, WU Xiao-Peng 3#, SONG Xin-Ming 1, HAN Chang-Ri 1, CHEN Zhong 2*, CHEN Guang-Ying 1* 1

Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158,

China; 2

College of Life Sciences, Hainan Normal University, Haikou 571158, China;

3

Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China Available online 20 Apr. 2014

[ABSTRACT] AIM: In an effort to identify novel, small molecules which can affect the proliferation of lung cancer cells, F-01A, a polyether antibiotic isolated from the fermentation broth of Streptomyces was tested. METHOD: F-01A was tested for its antitumor properties on the lung cancer cell line SPC-A-1, at six doses (0.1, 0.5, 1, 2.5, and 5 μmol·L−1), using various cellular assays. Cell viability was measured by the MTT assay, Hochest 33258 was used to study nuclear morphology; DNA ladder and the loss of mitochondrial membrane potential were also evaluated. RESULTS: F-01A induces apoptosis against SPC-A-1 cells in a dose-dependent manner. The IC50 is 0.65 μmol·L−1, and the inhibition at 5 μmol·L−1 is 87.89%. Further, JC-1 staining indicates F-01A could induce the loss of mitochondrial membrane potential, and the DNA fragment is evident. CONCLUSION: Mechanistic analysis showed that F-01A induced apoptosis of cancer cells probably in the mitochondrial pathway. The antitumor actions of F-01A involve activation of the apoptotic pathway against SPC-A-1 cells, and it may be valuable for further drug development. [KEY WORDS] Apoptosis; Membrane potential; SPC-A-1 cells; Polyether antibiotic

[CLC Number] R965

[Document code] A

[Article ID] 2095-6975(2014)04-0284-06 products with activities had been found from bacteria, such as Pseudomonas aeruginosa [2], Salmonella species [3], yeast [4], and Streptomyces species [5]. Some of the natural products are antibiotics, which exhibit broad spectrum, antibacterial activities and stronger antitumor activities towards many human tumor cell lines. A new bleomycin derivative NC0604 [6] was isolated from the fermentation broth of Streptomyces verticillus var. pingyangensis n.sp, which could increase the species of intracellular reactive oxygen and arrest human hepatoma HepG2 cells at G2/M phase. It also could activate rotein expression, such as p53 and Bax. Amphotericin B [7], a significant antifungal drug, was applied in AIDS-related fungal infections, in neutropenic cancer patients who are persistently febrile, and in infections of the central nervous system, lungs, peritoneum, genito-urinary system, eyes, and skin. The group of polyether natural products is of special interest owing to their fascinating structure and biological effects displayed by its members, which ranges from therapeutic antibiotic, antifungal [8], and anticancer properties [9], and extreme lethality. Some of the polyethers could induce

Introduction Natural products have played a highly significant role over the years in the discovery of new drugs. This is particularly evident in the treatment of cancer and infectious diseases in which more than 60% and 75% of drugs, respectively, are of natural origin [1]. Therefore, a natural product with strong synergistic activity, and minimal toxicity would be a new tool for cancer therapy. Many natural [Received on] 28-Nov.-2012 [Research funding] This project was supported by the National Nature Science Foundation of China (Nos. 31360069, 21162009) and the Program for New Century Excellent Talents in University (No. NCET- 08-0656). [*Corresponding author] CHEN Zhong: Prof., Tel/Fax: 86-89865889422, E-Mail: [email protected]; CHEN Guang-Ying: Prof., [email protected] # Co-first author These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

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the apoptosis of cancer cells [10]. Accumulating evidence shows that induction of apoptosis plays a very important role in tumor chemotherapy. Both extrinsic and intrinsic apoptotic signaling pathways are triggered by many antitumor agents [11-12]. The most relevant biological interest in apoptosis is the possibility of its modulation, hence, the identification of inductive and protective factors and their mechanisms of action seem to be the most relevant challenges in apoptosis research. The increasing number of studies enforcing the importance of mitochondria in apoptosis signaling, with an increase in complexity, has resulted in a strong debate concerning the exact sequence of mitochondrial events. Mitochondrial dysfunction in apoptosis is related with specific permeabilization of the outer mitochondrial membrane to large molecules, including ions that are relevant in the apoptotic process [13]. The detection of the mitochondrial permeability transition event provides an early indication of the initiation of cellular apoptosis. This process is typically defined as a collapse in the electrochemical gradient across the mitochondrial membrane, as measured by the change in the mitochondrial membrane potential.

(Beyotime, Hangzhou, China) were used. Ultraviolet (UV) absorbance was measured with Microplate reader EL×800 (BioTek, VT, USA). 5-Fluorouracil was used as the positive control (Sigma, St. Louis, MO, USA). Assays to test the apoptosis of SPC-A-1 induced by F-01A using the MTT assay The cell proliferation was evaluated using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay [16]. The cells were incubated in a 96-well microtitre plate with 180 μL/well at a proper density (i.e. 2 × 104, 7 × 104, 3 × 104 and 1 × 104 cells per micro liter medium for SPC-A-1, BEL-7402, GSC-7901, and K562, respectively), and then treated with 20 μL of various concentrations of F-01A and 5-fluorouracil (5-Fu) at the indicated times. Next, 50 μL of MTT (Sigma) reagent solution (5 mg·mL−1) was added and incubated for 4 h. After the medium and MTT were removed, 150 μL of DMSO (Tianjing, China) were added, then placed on a plate shaker for 5 min at room temperature. Cell viability was determined according to the manufacturer's instructions. Cell survival rate was calculated as the percentage of MTT inhibition as follows: percentage of survival = (mean experimental absorbance/mean control absorbance) ×100%. Nuclear morphology study by Hoechst 33258 To observe the nuclear changes occurring during apoptosis, the chromatin-specific dye Hoechst 33258 was used [17]. SPC-A-1 cells were plated into a 6-well plate at a density of 2 × 106 cells/well. Then they were exposed to F-01A (0, 0.1, 0.5, 1, 2.5, 5 μmol·L−1, 5-Fu 0.5 mmol·L−1) for 24 h, stained with Hoechst dye (10 μg·mL−1) for 30 min. After washing with PBS, the cells were analyzed under a fluorescence microscope (Nikon, Tokyo, Japan) and photographed. DNA ladder assay detection of the apoptosis F-01A-induced apoptosis in SPC-A-1 cells was determined in the form of fragmented DNA following the method of Laird et al [18]. Briefly, after treating SPC-A-1 cells without or with F-01A at desired concentrations and time intervals, cells were harvested, washed with PBS (pH 7.4) and incubated with lysis buffer [10 mmol·L−1 Tris-HCl (pH 8.5), 5 mmol·L−1 EDTA, 0.2% SDS, 0.2 mol·L−1 NaCl, 0.1 mg·mL−1 proteinase K] at 37 °C for 2 to 3 h. DNA was extracted by mixing an equal volume of isopropanol to the lysate. The lysate was then centrifuged at 3 000 r·min−1 for 5 min to pellet down the DNA. The pelleted DNA was air-dried and re-suspended in 500 AL of 10 mmol·L−1 Tris-HCl, 0.1 mmol·L−1 EDTA (pH 7.5) for complete dissolution overnight at 55 °C. DNA (1 mg) was resolved over 1% agarose gel following gel electrophoresis. The gel was then stained with 1 μg·mL−1 of EB in 1-tris-borate-EDTA buffer. The bands were visualized under a UV transilluminator followed by Polaroid photography. Mitochondrial permeability potential Cells were stained with the cationic dye JC-1 (Beyotime,

Materials and Methods Preparation of F-01A F-01A, isolated from the fermentation broth of Streptomyces, is a polyether compound (Fig. 1). It was supplied by the Institute of Tropical Bioscience and Biotechnology (Haikou, China), and separated by Xiaopeng Wu. According to the literature, it can be used as an antibiotic [14-15]. And HPLC analysis of purity at 201 nm was 99.3%.

Fig. 1 Structure of F-01A

Cells and chemicals Four cancer cells (SPC-A-1, human lung cancer cell Line; BEL-7402, human hepatocellular carcinoma cell line; SGC-7901, human gastric cancer cell line; and K562, human myelogenous leukemia cell line) were originally obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM medium (Sigma, St. Louis, MO, USA) containing 10% neonatal bovine serum (NBS, Hangzhou, China). The cells were maintained at 37 °C and 5% CO2. For experiments, the cells were seeded in 96-well culture plates and grown in complete medium to 90% confluence. Then, the cells were washed with phosphate-buffered saline (PBS) and incubated for 48 h at 37 °C in 1 980 μL of medium containing F-01A. Caspase 3 Activity Assay Kit and Cell Cycle and Apoptosis Analysis Kit

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incubated them at 37 °C for 20 min in a CO2 incubator and, after discarding the medium, was washed twice with JC-1 solution (1 mL). The wash was discarded and a drop of culture medium was added to the wells prior to immediate examination in the fluorescence microscope. Statistical analysis Data are presented as means ± SD. Statistical analysis was performed by one-way analysis of variance (ANOVA) using SPSS 11.0 software for Windows and P < 0.05 was considered statistically significant.

China), which exhibits potential-dependent accumulation in mitochondria. At low membrane potentials, JC-1 continues to exist as a monomer and produces a green fluorescence (emission at 527 nm). At high membrane potentials or concentrations, JC-1 forms aggregates (emission at 590 nm) and produces a red fluorescence. Cells were stained as recommended by the suppliers. Briefly, cells were cultured with various concentrations of F-01A in 6-well plates for 24 h, and after discarding the culture medium, culture medium (1 mL) and staining solution (1 mL) were added to the wells. Then

Table 1 The inhibition and IC50 of F-01A against four human cancer cell lines Cancer cell line SPC-A-1 SGC-7901

% Inhibition −1

0.1 μmol· L 22.56 0

−1

1 μmol·L

−1

5 μmol·L

−1

10 μmol·L

−1

50 μmol·L

−1

100 μmol·L

IC50/ (μmol·L−1)

IC50 of 5-Fu /(mmol·L−1) 0.5

34.35

87.89

94.62

95.88

96.17

0.65

30.82

97.01

97.39

97.85

99.27

3.27

0.08

74.55

2.34

0.1

2.11

0.2

K562

46.29

72.67

72.79

74.03

74.05

BEL-7402

0.34

9.31

87.43

98.69

99.52

Results F-01A treatment inhibits cellular proliferation of four human cancer cell lines To assess the antiproliferative effects of F-01A in SPC-A-1 cells, the dose-dependent effect was determined by the MTT assay. It was observed that in vitro treatment of F-01A resulted in antitumor effects on the human lung cancer SPC-A-1 cells (Table 1). Doses of more than 5 μmol·L−1 of F-01A, completely inhibited the proliferation potential of cells, and the IC50 was the smallest against SPC-A-1 cells at 0.652 μmol·L−1. From these results, the SPC-A-1 cell line was chosen to study the apoptosis mechanism of F-01A. The effect of F-01A on the cell viability of SPC-A-1 cells was further determined and confirmed by the MTT assay. According to this experiment, it was found that doses of 5 μmol·L−1 and upward of F-01A resulted in significant inhibition of cell viability. Therefore, in further mechanistic studies, doses of 0.1, 0.5, 1, 2.5, and 5 μmol·L−1 of F-01A were used. Cell viability of SPC-A-1 cells by F-01A SPC-A-1 cells were treated with varying doses of F-01A for 24, 48, and 72 h (Fig. 2). Treatment of F-01A with 2.5 μmol·L−1 significantly reduced the cell viability of SPC-A-1 cells from 24 h onwards up to 72 h, whereas a 34% to 5% reduction in cell viability was observed at concentrations of 5 μmol·L−1 of F-01A at 24, 48 and 72 h of treatment. These observations indicated the antiproliferative and anticarcinogenic potential of F-01A against human lung cancer cells. F-01A also inhibited significantly the proliferation of SPC-A-1 cells in a dose- and time-dependent manner. F-01A treatment induces apoptosis in SPC-A-1 cells The study was extended to examine whether the lung cancer cells are undergoing apoptosis after F-01A treatment. Induction of apoptosis in SPC-A-1 cells caused by F-01A treatment was assessed by using the DNA ladder assay. The data indicated that treatment of F-01A (0.1−5 μmol·L−1) to SPC-A-1 cells for 24 h markedly damaged or fragmented

100

DNA molecules in cancer cells, as evident by DNA ladder formation assay (Fig. 3). In further experiments, the F-01A-induced apoptosis in SPC-A-1 cells was morphologically identified using fluorescence staining with Hoechst 33342, as shown in Figs. 4A−F. Cells were treated without F-01A (Fig. 4A) and with F-01A (Figs. 4B−E) and 5-Fu (Fig. 4F), for 24 h. Fluorescence microscopy data indicated that F-01A treatment dose- and time-dependently induced apoptosis in lung cancer cells. In these observations, fragmented nuclei were observed in F-01A treated cells, as evident in Fig. 3. It was also interesting to quantify and verify the number of apoptotic cells induced by F-01A treatment to SPC-A-1 cells.

Fig. 2 Determination of SPC-A-1 cell line proliferation by the MTT assay. *P < 0.05, **P < 0.01 vs control group

Fig. 3 DNA electrophoresis of SPC-A-1 cells after treatment with F-01A for 24 h. (Left: DNA Ladder, control, 0.5, 1, 2.5, 5 μmol·L−1 F-01A, and 5-Fu)

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Fig. 4 Hochest 33258 staining map of control group cells. Concentration of F-01A: 0 μmol·L−1 (A); 0.1 μmol·L−1 (B); 0.5 μmol·L−1 (C); 1 μmol·L−1 (D); 2.5 μmol·L−1 (E); and 5 μmol·L−1 (F)

Exposure of SPC-A-1 cell line to F-01A to led to collapse in mitochondrial transmembrane potential The maintenance of mitochondrial membrane potential is significant for mitochondrial integrity and bioenergetic function [19]. Mitochondrial changes, including loss of Δψm, are key events that take place during drug-induced apoptosis. To determine the changes in Δψm, a unique fluorescent cationic dye, 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl benzimidazolylcarbocyanine iodide, commonly known as JC-1, which is specific for mitochondria was used. JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (λ 514 nm) to red (λ 585 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. The potential-sensitive color shift is due to the concentrationdependent formation of red fluorescent ‘‘aggregates’’ [20]. Therefore, a careful analysis of the fluorescence ratio detected allows for comparative measurements of membrane potential to be made, and a determination of the percentage of mitochondria within a population that responds to an applied stimulus [21-24]. As shown in Figs. 5 A−G, with the increasing concentration, the red fluorescence was reduced and the green fluorescence increased. Some cells were green at λ 514 nm, but missed at λ 585 nm, which means the mitochondrial membrane potential had collapsed.

Fig. 5 JC-1 fluorescence imaging of mitochondria in various concentration group cells. Concentration of F-01A: 0 μmol·L−1 (A); 0.1 μmol·L−1 (B); 0.5 μmol·L−1 (C); 1 μmol·L−1 (D); 2.5 μmol·L−1 (E); 5 μmol·L−1 (F). Positive control: 5-Fu (G). (Left: λ 514 nm, Right: λ 585 nm)

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Discussion

[2]

Apoptosis has been characterized as a fundamental cellular activity, and plays a crucial role as a protective mechanism against neoplastic development in the organisms by eliminating genetically damaged cells or those cells that have been improperly induced to divide by a mitotic stimulus [17, 25]. Acquired resistance toward apoptosis is a hallmark of most, and perhaps all, types of cancer. The balance between apoptosis and anti-apoptosis signaling pathways plays a role in the pathogenesis of a variety of cancers. It has been demonstrated that the inhibition of apoptosis promotes the mitotic progression in cancer cells [26]. Tumor development and progression as well as resistance to most oncologic therapies result mainly from a lack in the response to apoptotic stimuli [27]. The mechanisms of apoptosis induced by natural product are diverse, such as active caspase-3 and -7 [28], increasing the level of p53 protein and downgrade Bcl-2 protein [29], producing reactive oxygen species (ROS) [30], and disrupting the mitochondrial membrane potential [31], among thers. Due to metabolic and morphological similarities, monocytic SPC-A-1 cells have been accepted as a good model of lung carcinoma. F-01A is a polyether antibiotic, and was separated from the fermentation broth of a Streptomyces strain and many antibiotics have been found to induce apoptosis in cancer cells [32-34]. In the present study, F-01A significantly suppressed the proliferation of SPC-A-1 cells. Moreover, the nuclear morphology and JC-1 staining suggested that an apoptotic cell death mechanism was also potentially involved in this direct apoptosis. Because F-01A could inhibit growth and induce G1 arrest and apoptosis in SPC-A-1 cells, attention was paid to its potential mechanism. F-01A may regulate apoptosis by directly increasing the activity of caspases, by mainly upgrading the activity of caspase-3 [35]. In the present study, it was found that the F-01A-induced apoptosis of SPC-A-1 cells was also related to the loss of mitochondrial membrane potential and is consistent with the data in Figure 3B-F which shows the appearance of apoptotic cells. However, the active pharmacophore in F-01A that affects apoptosis in the SPC-A-1 cells needs to be characterized in more detail. In summary, it was shown that F-01A, which is a well-characterized and well-established compound, induces apoptosis of SPC-A-1 cells through nuclear degradation, and lose mitochondrial membrane, which may provide a molecular explanation for its potent in vitro anticancer properties. In addition, the effects of F-01A treatment on apoptosis in SPC-A-1 need to be characterized in more detail, such as the density of caspase-3, p53 protein, Bcl-2 protein, and ROS, which will be studied subsequently.

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Cite this article as: WANG Jing, WU Xiao-Peng, SONG Xin-Ming, HAN Chang-Ri, CHEN Zhong, CHEN Guang-Ying. F-01A, an antibiotic, inhibits lung cancer cells proliferation [J]. Chinese Journal of Natural Medicines, 2014, 12(4): 284-289

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