Jin-hui Shao* and Gui-hua Feng

1Department of Medical Morphology, School of Medicine, Hubei University of Art and Science, Xiangyang 441053, Hubei, China

2Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China

Inhibition Mechanism of Novel Pyrazolo[1,5-a]pyrazin-4(5H)-one Derivatives Against Proliferation of A549 and H322 Cancer Cells△

Jin-hui Shao1,2* and Gui-hua Feng1

1Department of Medical Morphology, School of Medicine, Hubei University of Art and Science, Xiangyang 441053, Hubei, China

2Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China

pyrazole； apoptosis； lung cancer cell； p53； human umbilical vein endothelial cell

Objective To explore the inhibition mechanism and safety of pyrazolo［1，5-a］pyrazin-4（5H）-one derivatives against proliferation of human lung cancer A549 cells， H322 cells， and human umbilical vein endothelial cell（HUVEC）.

Methods Cells were treated with 40 μmol/L of the ppo3a， ppo3b， ppo3i， and 0.1% DMSO （control）for 48 hours， respectively. Apoptosis was determined by Hoechst 33258 staining assay in H322 and A549 cells. Cell cycle distribution was determined by flow cytometry analysis in A549 cell. LC3-II， p53， and heat shock protein （HSP） 70 protein levels were detected by Western blotting in A549 cells treated with ppo3b for 48 hours. The morphology and viability of HUVEC were observed by inverted microscope and sulforhodamine B（SRB） assay.

Results Ppo3a， ppo3b， and ppo3i significantly induced apoptosis in H322 and A549 cells. A strong G1-phase arrest was concomitant with the growth inhibitory effect on A549 cells. Ppo3b effectively elevated the p53 protein level， but significantly reduced the HSP70 protein level. There were no significantly inhibitory effect on the morphology and viability of HUVEC when treated with ppo3a，ppo3b， and ppo3i.

Conclusions ppo3a， ppo3b， and ppo3i could inhibit H322 proliferation through apoptosis and inhibit A549 through apoptosis and G1-phase arrest. The protein p53 and HSP70 might involve in the inhibition effects. These derivatives might be a clue to find effective and safe drug for lung cancers.

Chin Med Sci J 2015； 30（4）:260-265

CANCER is a major public health problem worldwide. Lung cancer， which is one of the most common malignant human tumors， is a leading cause of death worldwide.1A549 cell is human lung adenocarcinoma cell line， as such， they are the best model for lung cancer studies. A key feature of cancer cells is uncontrolled proliferation. Thus， inhibition of proliferative pathways may be an effective strategy in fighting cancer. Chemotherapy remains one of the primary modalities for cancer treatment.2，3

A number of pyrazole derivatives exhibit a wide range of biological properties， including anticancer bioactivity. Considerable work has been directed toward the design and synthesis of fused-pyrazole derivatives.4，5In our previous papers，6a series of novel pyrazolo ［1，5-a］ pyrazin-4（5H）-one derivatives were synthesized， and preliminary biological evaluation was performed. The results showed that the compounds can inhibit the growth of A549 and H322 cells in a dose-dependent manner. Among the derivatives，ppo3a， ppo3b， and ppo3i can significantly inhibit the proliferation of A549 cells and H322 cells. The mechanism by which the proliferation of lung cancer cells was inhibited remains unknown， but not through necrosis. Thus， we performed further experiments to determine the mechanism. We analyzed the actions of pyrazolo［1，5-a］pyrazin-4（5H）-one derivatives on human lung cancer A549 cells and H322 cells to determine its possible inhibitory activity against proliferation of culture cells. Meanwhile， the safety of the compounds was also evaluated using human umbilical vein endothelial cell （HUVEC）.

MATERIALS AND METHODS

Cell culture and treatment

A549 and H322 lung cancer cells were cultured in RPMI 164O medium （Gibco BRL Co.， Grand Island， USA） at 37°C under 5% CO2and 95% air. The medium was supplemented with 1O% （v/v） bovine calf serum （Ding-Guo Biotechnology Co.， Beijing， China） and 8O U/ml gentamicin. The cells were seeded onto 96-well plates or 24-well plates containing the medium at a density of 6OOO cells/cm2. HUVECs were isolated from human umbilical veins in our laboratory by the method previously described.7The detached cells were cultured on gelatin-coated plastic dishes with the M199 medium （Gibco BRL Co.）， which was supplemented with 2O% fetal bovine serum and 7O ng/ml fibroblast growth factor-2 in 5% CO2at 37°C. The cells were seeded onto 96-well plates or 24-well plates containing the medium at a density of about 6OOO cells/cm2. Cells were treated with 4O μmol/L of the ppo3a， ppo3b， ppo3i， and O.1% dimethyl sulphoxide （DMSO， control） for 48 hours， respectively. Ppo3a， ppo3b， and ppo3i were donated by Professor Bao-xiang Zhao of Shandong University. DMSO was purchased from Shanghai Sangon Biological Engineering Technology and Services Company （China）.

Hoechst 33258 staining assay

For A549 and H322 cells， cell fixation was performed with 4% formaldehyde in PBS for 1O minutes before staining with 2 μg/ml of Hoechst 33258 at 37°C for 3O minutes. Afterward， the cells were gently washed once with PBS and were then observed under a fluorescence microscope（Nikon， Japan）. The condensed DNA of the apoptotic cells was identified by intense staining in the nucleus in contrast to diffused staining of DNA in normal cells. Apoptosis rate means the ratio of apoptotic cells to total counted cells. A minimum of 5OO cells was counted. Each experiment was performed in triplicate.8，9

Flow cytometry analysis

A549 cells were harvested after treated with O.25% trypsin and fixed in 7O% ethanol and then stained with 5O μg/ml propidium iodide （PI） containing 1O μg/ml RNase A at 4°C for 1 hour. The stained cells were analyzed using a FACS Calibur Flow Cytometer （BD Bioscience， USA）. The cell cycle distribution was determined using the ModiFit software （BD Bioscience， USA）.1O，11

Western blotting analysis

Cells were washed twice with ice-cold PBS and then lysed in a protein lysis buffer （O.5% SDS in 25 mmol/L Tris-HCl，pH 7.5， 4 mmol/L EDTA， 1OO mmol/L NaCl， 1 mmol/L PMSF， 1O μg/ml leupeptin， and 1O μg/ml soybean trypsin inhibitor）. The protein concentrations of the cells were determined by the Bradford method.12The equal amounts of proteins were loaded into 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and electrophoretically transferred onto a polyvinylidene fluoride membranes（Millipore， MA， USA）. The membranes were blocked with 5% non-fat milk in PBST （O.O5% Tween-2O） buffer for 1 hour at room temperature. Rabbit anti-human LC3Ⅱantibodies （1：5OO）， anti-p53 mouse monoclonal antibodies（1：2OO）， anti-heat shock protein （HSP） 7O mouse monoclonal antibodies （1：5OO）， or anti-GAPDH mouse monoclonal antibodies （1：5OO） were added and incubated overnight at 4°C. After washed twice with PBST， the membrane was incubated with the secondary antibody for 1 hour at room temperature and then washed three times with PBST. Afterward， the membrane was incubated with HRP substrate until the protein bands were appeared. Theintensities of the protein bands were quantified using the Image J software （National Institutes of Health， USA）.13

Antiproliferative activity

Proliferation percentage was determined by the sulforhodamine B （SRB） assay. The HUVECs were incubated with ppo3a， ppo3b， and ppo3i at the concentrations of 4O μmol/L for 48 hours， and the cell proliferation/viability was determined using the survival percentage with the cells treated only with DMSO at O.1% as a reference. The results were expressed as the average of triplicate assays.

Statistical analysis

Statistical analysis was performed with Quantity-One soft- ware（Bio-Rad）. Measurement data were presented as means ± SE. Comparisons of means between two groups were performed with student's t-test. Enumeration data were presented as ratio. P＜O.O5 was considered statistically significant.

RESULTS

Compound-induced apoptosis in H322 cells and A549 cells

Hoechst 33258 staining showed that chromatin condensation and DNA fragmentation were appeared in H322 cells and A549 cells （Fig. 1A）. Compared with the control group， the apoptosis rates of ppo3a， ppo3b， and ppo3i groups were significantly increased （all P ＜ O.O5） （Fig. 1B）.

Cell cycle distribution of A549 cells

Flow cytometry results showed that ppo3a， ppo3b， and ppo3i effectively induced G1-phase arrest of A549 cells （Fig. 2）. Compared with the control group， G1 population noticeably increased in ppo3a， ppo3b， and ppo3i by 3O%， 31%， and 32%， respectively. The increases in the G1-phase cell populations were accompanied by decreasing in the S- and G2-phase cell populations. The strong inducement of the G1-phase arrest by ppo3a， ppo3b， and ppo3i were concomitant with their growth inhibitory effects.

LC3-II, p53, and HSP70 expression in A549 cells

According to the result of Western blotting， ppo3b effectively increased the p53 protein level （1.8O±O.O7， P= O.OO7）and significantly decreased the HSP7O protein level in A549 cells （O.61±O.O6， P= O.O24）. However， there was no significantly difference in LC3-II expression between the ppo3b and control groups （O.98± O.O4， P = O.665） （Fig. 3）.

Effects of the compounds on HUVEC viability and morphology

After incubated with 4O μmol/L ppo3a， ppo3b， and ppo3i for 48 hours respectively， the viabilities of the HUVECs were not obviously suppressed. Meanwhile， no significant changes in morphology were observed in the treatments and control groups （Fig. 4）.

Figure 1. Ppo3a， ppo3b， and ppo3i-induced apoptosis in H322 and A549 cells.

Figure 2. Effects of ppo3a， ppo3b， and ppo3i on the cell cycle distribution of A549 cells.

Figure 3. Effect of ppo3b on LC3-Ⅱ， p53， and HSP7O protein expressions in A549 cells.

Figure 4. Morphologies of human umbilical vein endothelial cells treated with O.1% （v/v） DMSO （a）， 4O μmol/L ppo3a （b）， ppo3b（c）， and ppo3i （d） respectively for 48 hours. ×2OO

DISCUSSION

Three pyrazolo［1，5-a］pyrazin-4（5H）-one derivatives，namely， ppo3a， ppo3b， and ppo3i significantly induced apoptosis in H322 and A549 cells. While， those apoptosis rate of H322 cells were higher than those of A549 cells. The compounds can also induce strongly the G1-phase arrest of A549 cells.

Microtubule-associated protein 1 LC3-Ⅱ （MAP1 LC3-Ⅱ），the first autophagy membrane protein found in advanced eukaryotic cells， is a marker of autophagy. An increased level of LC3-Ⅱ expression generally indicates that autophagy is induced.14，15Treatment with ppo3b showed no significant effect on LC3-Ⅱ protein levels， which indicated that autophagy was not affected.

The p53 tumor suppressor gene ensures the genetic stability of multicellular organisms. This gene suppresses the accumulation of mutations in somatic cells and substantially reduces the probability of malignant diseases.16，17In our study， ppo3b effectively increased the level of p53 protein （P＜O.O1）， indicating that the antitumor pathway of ppo3b might be via p53 activation.

HSP7O， a highly conserved chaperone protein that refolds misfolded proteins， performs numerous housekeeping functions. Given the ability of HSP7O to protect cells from a wide range of apoptotic and necrotic stimuli，this protein is believed to confer survival advantage to tumor cells.18，19In the present study， ppo3b significantly decreased the HSP7O protein level （P＜O.O5）， indicating that ppo3b might through suppressing the expression of HSP7O and thus inhibited tumor growth.

No significant changes in morphology or quantity were observed after the treatment of the compounds on HUVEC. These showed that the antitumor effect of the compounds was selective and they were safe to normal cells. The use of available chemotherapeutics is often limited， mainly because of undesirable side effects as well as the limited choice of available anticancer drugs. Clearly， selective anticancer agents with higher and more potent antitumor activities must be developed.

Acknowledgments

I would like to thank Professor Jun-ying Miao and Professor Bao-xiang Zhao of Shandong University for the compounds and technical platform.

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for publication October 29, 2014.

Tel: 86-710-3591126, E-mail: jhshao666@163.com

△Supported by the Doctoral Research Fund of Hubei University of Art and Science (2013B009) and the Post-doctoral Research Fund of Shandong University (111935).