European Review for Medical and Pharmacological Sciences 2018; 22: 1323-1332 Wnt3 knockdown sensitizes human non-small cell type lung cancer (NSCLC) cells to cisplatin via regulating the cell proliferation and apoptosis

Z. XING1,2, H.-Y. WANG1,3, W.-Y. SU2, Y.-F. LIU1, X.-X. WANG1,4, P. ZHAN5, T.-F. LV1,5, Y. SONG1,5

1Department of Respiratory Medicine, Jinling Hospital, Southern Medical University, Nanjing, Jiangsu, China 2Department of Oncology Medicine, Inner Mongolia Medical University Affiliated Hospital, Hohhot, Inner Mongolia Autonomous Region, China 3Department of Infections Medicine, Inner Mongolia Medical Chifeng Hospital, Chifeng, Inner Mongolia Autonomous Region, China 4Intensive Care Unit, Inner Mongolia People’s Hospital, Hohhot, Inner Mongolia Autonomous Region, China 5Department of Respiratory Medicine, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu, China

Ze Xing and Hongying Wang contributed equally to this work

Abstract. – OBJECTIVE: Aberrant activa- Key Words: tion of (Wingless and mouse homolog Int-1) Lung carcinogenesis, Lung cancer, Wnt/β-catenin, Wnt/β-catenin signaling pathways closely in- siRNA, Invasion. volved in the occurrence and progression of several types of human malignancies. This re- search was undertaken to elucidate the import- Introduction ant role of (Wingless and mouse homolog Int-1) in lung cancer. PATIENTS AND METHODS: Lung cancer is the second common cancer Wnt3 expression 1 in lung cancers and their respective normal tis- type for both men and women worldwide or is sues were examined by immunoblotting and im- the most common one for men and the second munohistochemistry. Then, Wnt3 was regulated common one for women in China2. Moreover, with RNA interference (RNAi) technology in hu- it is markedly leading the mortality among all man lung cancer A549 cells, and the cell prolif- types of cancers both in China and worldwide1,2. eration, cell cycle, cell invasion/metastasis, and More than 80% lung cancers are histological- apoptosis were evaluated. RESULTS: In all cases, Wnt3 expression was ly classified into non-small cell type lung can- significantly elevated in lung cancers compared cers (NSCLC), which includes adenocarcinoma with normal tissues. Knocking down Wnt3 in (ADC), squamous cell carcinoma (SCC), large A549 lung cancer cells by small interfering RNAs cell carcinoma (LCC) and other types3. Multiple transfection led to a distinct reduction of Wnt3 factors contributed to lung cancer development. in both transcript and levels. Knockdown Except for the family history4, the susceptibility of Wnt3 expression in lung cancer cells inhib- 5 ited the expression of β-catenin and cyclin D1 locus (such as 6q23-25) and genet- in Wnt/β-catenin pathway. It also signifi- ic polymorphisms (three main susceptibility loci, cantly blocked cellular proliferation, delayed cell 15q256, 5p157, and 6p218 regions) were associated cycle and suppressed cell invasion/metastasis, with lung cancer. Moreover, the epidemiological accompanied by a higher apoptosis rate. evidence and biological plausibility unanimously CONCLUSIONS: We conclude that the upreg- support a causal association between the tobacco ulation of Wnt3 plays a crucial role in lung tum- smoking and lung cancer risk9,10. There were sev- origenesis by inducing proliferation, migration, and invasion and inhibiting apoptosis of can- eral factors explaining the high mortality rate of cer cells. Wnt3 might be a potential target for the lung cancer, including late diagnosis, restriction treatment of lung cancer. on local eradication. More importantly, the cancer

Corresponding Author: Yong Song, MD; e-mail: [email protected] 1323 Z. Xing, H.-Y. Wang, W.-Y. Su, Y.-F. Liu, X.-X. Wang, P. Zhan, T.-F. Lv, Y. Song associated biological factors and biological mech- in liquid nitrogen and stored at -80°C before use. anisms underlining the aggressiveness, the tumor Detailed clinical and pathological information heterogeneity and the acquisition of resistance of about these patients were listed in Table I. lung cancer contributed to the bad outcome of lung cancer patients11. Immunohistochemical Staining for Wnt3 Wnt (wingless and mouse homolog Int-1)12 NSCLC tissues were fixed with 4% formal- family encodes a group of evolutionari- dehyde for the preparation of histopathologic ly-conserved glycoproteins, which modulate slide. For the immunohistochemical staining embryonic development, tissue regeneration, for Wnt3, slides were bathed in citrate buffer and many other processes13. The aberrant for 15 minutes for antigen retrieve. The Wnt3 Wnt/β-catenin signaling has been recognized detection was sequentially performed with the during the onset and progression of various first incubation with rabbit anti-human Wnt3 types of cancers, such as colorectal cancer14, antibody (Abcam, Cambridge, UK) for one gastric cancer15, breast cancer16 and others. In hours at 37°C, with the secondary incubation particular, various members of Wnt family are with the HRP-linked anti-rabbit antibody (Cell indicated to be frequently upregulated in lung Signaling Technology Inc., Danvers, MA, USA) cancers17-19. As a member of Wnt family, Wnt3 is for 30 minutes at room temperature and, then, also relevant to the hepatic20, lung19,21, gastric15, with the Mayer’s hematoxylin staining for the and colorectal carcinogenesis22. Moreover, the specific Wnt3-antibody binding. upregulated promotes tumor progression of NS- CLC, is significantly associated with a reduced Reagents, Cell Culture, and Treatment overall survival rate of NSCLC patients19. How- Human NSCLC A549 cells were purchased ever, the mechanism underlining the oncogenic from American Type Culture Collection (ATCC role of Wnt3 in NSCLC was not yet clear. Manassas, VA, USA), and were cultured in In the present study, we examined theWnt3 in the Dulbecco’s modified Eagle’s medium expression in NSCLC tissues. Then, we knocked (DMEM) (Gibco, Grand Island, NY, USA), down Wnt3 with RNA interference (RNAi) adding 10% FBS (GE Healthcare Life Sciences, technology, and investigated the regulatory role HyClone Laboratories, South Logan, UT, USA) of Wnt3 on the cell proliferation, cell cycle, and at 37°C in a humid incubator with 5% CO2. apoptosis. Our findings indicated that the over- Cells with approximately 85% confluence were expressed Wnt3 in NSCLC mediated (at least in splitt and were updated with fresh medium. For part) lung carcinogenesis by promoting cellu- the Cisplatin treatment, 2 μM Cisplatin (Sig- lar proliferation and reducing cell apoptosis and ma-Aldrich, St. Louis, MO, USA) was added might be a potential therapeutic NSCLC target. into the cells’ medium.

Patients and Methods Table I. Clinico-pathological features of NSCLC patients Collection of Non-small Cell Type Lung in this study. Cancers (NSCLC) Specimens Features Cases The study was approved by the Ethics Com- mittee of Jinling Hospital, Southern Medical Uni- Age at diagnosis versity. The written consent form was obtained < 55 9 ≥ 55 21 from all the participants. Human NSCLC tissue Gender samples (n = 30) and paired para cancer samples Male 16 (more than 1 cm away from the edge of tumor) Female 14 were collected during surgery on the NSCLC Tumor size (cm) patients, who underwent surgical eradication, < 13 ≥ 17 before any chemotherapy, radiation therapy or Tumor stage adjunctive therapy, at our hospital between Nov I+II 17 2014 and Jul 2015. The patients were selected ac- III 13 cording to clinicopathological characteristics (see Lymph node metastasis No 16 details in Table I). Each sample was immediately Yes 14 fixed with 4% formaldehyde or was snap-frozen

1324 Wnt3 regulates the proliferation and apoptosis of NSCLC cells

Quantitative Real-time Polymerase post the transfection with Wnt3-specific siRNA Chain Reaction (PCR) Analysis of Wnt3 (siRNA 1/2-Wnt3) or with control siRNA (siR- mRNA Level NA-Ctrl) for 0, 24, 48 or 72 hours. The 450 nm mRNA samples from NSCLC tissues or absorbance of each cell well was detected with A549 cells were prepared with the TRIzol re- a spectrophotometer (Crystaleye, Olympus, agent (Life Technologies, Grand Island, NY, Tokyo, Japan). The colony formation assay of USA), and were supplemented with RNase A549 cells was performed as follows. 200A549 inhibitor (Thermo Scientific, Waltham, MA, cells were seeded in 12-well plate, and then USA) before use. The quantitative analysis was were transfected with 50 nM siRNA 1/2-Wnt3 performed with TaqMan® Fast Advanced Mas- or siRNA-Ctrl. The cells were incubated for ter Mix kit (Thermo Scientific, Waltham, MA, further 72 hours. The cell colonies were stained USA) with Wnt3 specific primers (Forward: with 0.007% crystal violet for 10 minutes and 5’-AGAGAAGCACCGTGAGTCCCG-3’, Re- were imaged with UVP BioSpectrum 500 im- verse: 5’-GTGGGTGGCTTGAAGAGCG-3’) aging system (Upland, CA, USA). and probe (5’-FAM-TGGGTGGAGACCCTC- CGGGC-TAMRA-3’). The reaction was per- formed on the LightCycler 2.0 (Roche Diagnos- FACScan Flow Analysis for Cell Cycle tics, Mannheim, Germany). Primers and probes and Cell Apoptosis were synthesized by Sangon Biotech (Shanghai) For cell cycle assay, A549 cells post-treatment Co. (Shanghai, China). The Wnt3 level was pre- were digested with 0.25% EDTA-free trypsin sented as a fold change to the internal control and were resuspended in 700 μL binding buffer Glyceraldehyde 3-phosphate dehydrogenase supplemented with 5 μL7-amino-actinomycin (GAPDH) with the ∆∆Ct method23. (7AAD) for incubation at 37°C for 10 minutes. Then, another 300 μL binding buffer supple- Protein Isolation and Western mented with 2 μL annexin-V-phycoerythrin Blotting Assay were added. The stained A549 cells were fixed Protein samples from NSCLC tissues or A549 with 70% ethanol at 4°C for two hours, and then cells were lysed with a Cell Lysis Buffer (Cell were incubated with 0.5 mg/mL RNase Aat Signaling Technology Inc., Danvers, MA, USA) 37°C for 10 minutes, and then with 5 μL propid- and were supplemented with a protease inhibitor ium iodide at room temperature for 30 minutes. cocktail (Roche Biochemicals, Basel, Switzer- Cell cycle status was analyzed with a FACS- land). Protein samples were firstly separated by can flow cytometer (Bio-Rad, Hercules, CA, the electrophoresis with 10% sodium dodecyl USA) under the guidance of the manufacturer’s sulphate-polyacrylamide gel electrophoresis recommendations. For the apoptosis analysis, (SDS-PAGE) gel, and secondly were transferred A549 cells were resuspended in 500 μL bind- onto a nitrocellulose membrane (Millipore, Bill- ing buffer supplemented with 5 μL 7AAD for erica, MA, USA). Then, the membrane was firstly incubation at room temperature for 15 minutes. blocked with 2% bovine serum albumin (BSA) at Before the analysis, another 450 μL binding 4°C overnight, secondly incubated with the rabbit buffer and 1 μL annexin-V-phycoerythrin were anti-human Wnt3 antibody (Abcam, Cambridge, added in sequence. After 15 minutes, cell apop- MA, USA) (with GAPDH antibody as control) for tosis was analyzed using flowcytometry, and one hours at 37°C, thirdly incubated with the horse data analysis was performed by using FlowJo radish peroxidase (HRP)-linked anti-rabbit anti- software (Tree Star, Ashland, OR, USA). body (Cell Signaling Technology Inc., Danvers, MA, USA) for 40 minutes at room temperature. Four-time washing with PBST was performed Statistical Analysis before each incubation. Finally, the targeted blots GraphPad Prism 6.0 software (GraphPad Soft- were visualized with the enhanced chemilumi- ware, La Jolla, CA, USA) was utilized for the sta- nescence (ECL) detection systems (Amersham tistical analysis. Quantitative data were presented Pharmacia Biotech, Amersham, UK). as mean ± SE. Statistical difference was evaluated with Student’s t-test or ANOVA test, which was Cell Proliferation, Colony Formation Assay followed by Bonferroni test for a post-hoc test. A The proliferation of A549 cells was curved p-value < 0.05 or less was considered statistically with CCK-8 (DOJINDO, Kumamoto, Japan), significant.

1325 Z. Xing, H.-Y. Wang, W.-Y. Su, Y.-F. Liu, X.-X. Wang, P. Zhan, T.-F. Lv, Y. Song

Figure 1. Upregulated Wnt3 expression in non-small cell type lung cancer (NSCLC) patients. A, Relative mRNA level of Wnt3 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Non-small cell type lung cancer (NSCLC) tissues (n = 30), with para cancer tissues as control (n = 30). B and C, Western blot analysis (B) and relative protein level (C) of Wnt3 in NSCLC or para cancer tissues (n = 5 respectively). D and E, Immunohistochemistry staining of Wnt3 in para cancer (D) or in NSCLC (E) tissues (n = 15 respectively). Statistical significance was calculated with paired Student t-test and was indicated in each graph.

Results and protein levels in NSCLC specimens (n = 30). Figure 1A demonstrated that the relative Wnt3 Wnt3 is Overexpressed in Non-small Cell mRNA level (to GAPDH) was significantly high- Type Lung Cancers (NSCLC) Tissues er in the 30 NSCLC specimens than in the 30 para To determine whether Wnt3 signaling is dereg- cancer specimens (p = 0.0018). The Western blot- ulated in the microenvironment of NSCLC, we ting results (Figure 1B and 1C) indicated that the examined the expression of Wnt3 in both mRNA protein level of Wnt3 was also markedly higher in

1326 Wnt3 regulates the proliferation and apoptosis of NSCLC cells

NSCLC tissues than in the para cancer tissues (n On the other side, the G0/G1 phase was upregulat- = 5 respectively, p = 0.0044). In addition, the im- ed, however, not yet significantly. We also inves- munohistochemical analysis was also performed tigated the impact of Wnt3 knockdown on the cell to confirm the protein level of Wnt3 in NSCLC apoptosis of A549 cells. Figure 4A demonstrat- tissues. It was shown in Figure 1D and 1E that ed that Cisplatin (2 μM) induced more apoptotic theWnt3 was found to be overexpressed in NS- A549 cells, with the Wnt3 knockdown by either CLC samples, compared to the lower levels in the siRNA 1-Wnt3 or siRNA 2-Wnt3, in comparison para cancer tissues with a statistical significance with the A549 cells without a Wnt3 knockdown (n = 15 respectively, p = 0.0037, Figure 1F). Taken (p < 0.01 respectively, Figure 4B). In addition, we together, Wnt3 is overexpressed in NSCLC, prob- analyzed the expression of apoptosis-associated ably exerting an oncogenic effect in the onset and markers in the A549 cells, with or without Wnt3 development of NSCLC. knockdown. The Western blotting results (Fig- ure 5A) demonstrated that more caspase 9 and Downregulated Wnt3 by Wnt3-siRNA caspase 3 were activated (cleaved form, Cleaved Inhibits the Proliferation of NSCLC A549 CASP 9 or Cleaved CASP 3) (Figure 5B and 5C). Cells Taken together, these results revealed that down- To investigate a possible oncogenic or tumor regulation of Wnt3 deregulates cell cycle and pro- suppressive role of Wnt3 in NSCLC cells, we ma- motes apoptosis in NSCLC A549 cells. nipulated the Wnt3 expression with Wnt3-specific siRNA transfection. As indicated in Figure 2A, the transfection with 30 or 60 nM Wnt3-specific Discussion siRNA (siRNA 1/2-Wnt3) markedly downregu- lated the Wnt3 mRNA level than the transfection Previous studies demonstrated that various with control siRNA (siRNA-Ctrl) (p < 0.01 re- members of Wnt family are frequently upreg- spectively). And the downregulation of Wnt3 was ulated in multiple types of human cancers. The also confirmed in the siRNA 1/2-Wnt3-transfect- Wnt signaling has been recognized to associate ed (60 nM) A549 cells (p < 0.05 or p < 0.01, Fig- with the NSCLC prognosis24. The overexpres- ure 2B). We then curved the growth of the siRNA sion of Wnt1 and Wnt5a have been identified to 1/2-Wnt3- or siRNA-Ctrl-transfected A549 cells. pose effects in the tumor biology in NSCLCs25,26. Figure 2C demonstrated that the siRNA 1-Wnt3- In the present study, the overexpression of Wnt3 or the siRNA 2-Wnt3-transfected A549 cells was confirmed in both mRNA and protein levels grew less efficiently than the siRNA-Ctrl-trans- by three types of methods (immunohistochemis- fected A549 cells (p < 0.01 or p < 0.001). To re- try, Western blotting, and real-time quantitative confirm the inhibition by the Wnt3 knockdown on PCR). Wnt3 was indicated as an important bio- the proliferation of A549 cells, we then performed marker for NSCLC patients. As one member of a colony-forming assay for the three groups of Wnt family, Wnt3 gene is located on human chro- A549 cells. Figure 2D indicated that there were mosome 17q2127, and its product stimulates the less colonies formed by the A549 cells, post the canonical Wnt/-catenin pathway27,28. The activat- transfection with 60 nM siRNA 1-Wnt3 (p < 0.01) ed Wnt/-catenin complex was accumulated in the or siRNA 2-Wnt3 (p < 0.05). cytoplasm29, was translocated into the nucleus and then activates the transcription of the Wnt-target Downregulated Wnt3 by Wnt3-siRNA genes. However, the mechanism underlining the Deregulates the Cell Cycle and Induces oncogenic role of Wnt3 remains not clear. the Apoptosis of NSCLC A549 Cells Wnt3 has also been recognized to promote the Further experiments were performed to exam- tumor progression in NSCLC30. Our followed re- ine the regulation by the Wnt3 knockdown on the sults also confirmed the promotive role of Wnt3 cell cycle distribution, in the presence of low level in the progression of NSCLC cells. The marked of Cisplatin (2 μM). Flow cytometry showed that knockdown of Wnt3 with RNAi technology in contrast to the siRNA-Ctrl-transfected A549 posed a significant regulation on the prolifera- cells (Figure 3A), less proportion of cells in S tion of NSCLC A549 cells. The growth curve of phase was observed in the groups of A549 cells, either siRNA 1-Wnt3- or siRNA 1-Wnt3-trans- post the siRNA 1-Wnt3 (Figure 3B) or siRNA fected A549 cells was much lower than the siR- 2-Wnt3 (Figure 3C) transfection, with a signifi- NA-Ctrl-transfected A549 cells and the colo- cant difference (p < 0.01 respectively, Figure 3D). nies formed by either siRNA 1-Wnt3- or siRNA

1327 Z. Xing, H.-Y. Wang, W.-Y. Su, Y.-F. Liu, X.-X. Wang, P. Zhan, T.-F. Lv, Y. Song

Figure 2. Proliferation of NSCLC A549 cells post the knockdown of Wnt3 with siRNAs. A, Relative mRNA level of Wnt3 to GAPDH in NSCLC A549 cells, which were transfected with 50 nM Wnt3-specific siRNAs (siRNA 1/2-Wnt3) or control siRNA (siRNA-Ctrl) for 24 hours. B, Western blot analysis of Wnt3 expression in protein level in A549 cells, which were transfected with 50 nM siRNA 1/2-Wnt3 or siRNA-Ctrl for 24 hours, with GAPDH as an internal control. C, CCK-8 analysis of the proliferation of siRNA 1/2-Wnt3- or siRNA-Ctrl-transfected (50 nM) A549 cells. D, Colony formation assay of siRNA 1/2-Wnt3- or siRNA-Ctrl-transfected (50 nM) A549 cells. Experiments were performed independently in triplicate. Statistical significance was shown as p* < 0.05, **p < 0.01 or ***p <0.001.

1328 Wnt3 regulates the proliferation and apoptosis of NSCLC cells

Figure 3. Cell cycles of the A549 cells post the knockdown of Wnt3 with siRNAs. A-C, Flow cytometry analysis of the cell cycle distribution in the A549 cells, which were transfected with 50 nM Wnt3-specific siRNAs (siRNA 1/2-Wnt3) or control siRNA (siRNA-Ctrl) for 24 hours, in the presence of 2 μM Cisplatin. D, The percentages of cells in the G0/G1, S, G2/M phases of the cell cycle in each group were measured by using the cellQuest Pro software.

1-Wnt3-transfected A549 cells were also less than tin-induced more apoptotic A549 cells, with the the colonies formed by siRNA-Ctrl-transfected Wnt3 knockdown, in comparison with the A549 A549 cells. Such role of Wnt signaling has also cells without Wnt3 knockdown. And our Western been confirmed in other studies in which inhibi- blotting results presented more activated caspase tion of Wnt/β-catenin signaling with Wnt inhibi- 9 and caspase 3 in the Wnt3 knocked-down A549 tor or Wnt depletion attenuated the proliferation cells. Therefore, Wnt3 knockdown sensitized and motility of NSCLC cells and increased their human non-small cell type lung cancer cells to apoptosis31. cisplatin via regulating the cell proliferation and The Wnt signaling has been recognized to as- apoptosis. More importantly, our results demon- sociate with the resistance of metastatic NSCLC strated that the Wnt3 knockdown also deregulat- to chemotherapy24,32. Our study indicated a sen- ed the cell cycle of A549 cells. Flow cytometry sitization effect by the Wnt3 knockdown. Cispla- results demonstrated a less proportion of cells in

1329 Z. Xing, H.-Y. Wang, W.-Y. Su, Y.-F. Liu, X.-X. Wang, P. Zhan, T.-F. Lv, Y. Song

Figure 4. Apoptosis in the A549 cells post the knockdown of Wnt3 with siRNAs. A549 cells were transfected with 50 nM siRNA 1/2-Wnt3 or siRNA-Ctrl, and then were incubat- ed with 2 μM Cisplatin for 24 hours; then, the cell apoptosis was assessed by flow cytometry using Annexin V-FITC/PI Apoptosis Detection Kit. A and B, Representative images (A) and the percentage (B) for apoptotic A549 cells, post the transfection with siRNA 1/2-Wnt3 or siRNA-Ctrl (Lat- er apoptotic cells were denoted in the upper right quadrant, and the early apoptotic cells were denoted in the lower right quadrant). **p < 0.01.

S phase while a more proportion of cells in G0/ G1 phase in the Wnt3 knockdown group than in the control group of cells. We speculated that the regulatory role of Wnt3 in the sensitization of NS- CLC cells to cisplatin might be associated with the deregulation by it on the cell cycle.

Conclusions

Wnt3 is overexpressed in non-small cell type Figure 5. Western blotting assay for apoptosis-associat- lung cancer tissues, Wnt3 knockdown sensitizes ed caspases in the A549 cells post the Wnt3 knockdown. A549 cells were transfected with 50 nM siRNA 1/2-Wnt3 NSCLC cells to chemotherapy, via inhibiting the or siRNA-Ctrl, and then were incubated with 2 μM Cis- proliferation, inducing the apoptosis and deregu- platin for 24 hours; then, Western blotting assay was per- lating the cell cycle of NSCLC cells. Our study formed to examine the levels of active caspase 9 and active suggests that Wnt3 signaling might be effective caspase 3 (Cleaved CASP 9 or Cleaved CASP 3). A, Rep- target for the chemotherapy of NSCLC. resentative Western blot analysis (A) and the relative levels of Cleaved CASP 9 (B) or Cleaved CASP 3 (C) in the siR- NA 1/2-Wnt3- or siRNA-Ctrl-transfected A549 cells, post the Cisplatin treatment (2 μM for 24 hours). Experiments Conflict of Interest were performed independently in triplicate. **p < 0.01, ns: The Authors declare that they have no conflict of interest. no significance.

1330 Wnt3 regulates the proliferation and apoptosis of NSCLC cells

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