MP06 (LAV ISW KCQ EWN SLW KKR KRK T-NH2) and FITC-MP06 (with FITC tagged at the N-terminal) peptides (>95% purity) were synthesized by DANDI Cure Co. (Republic of Korea) through a solid-phase synthesis method. The molecular masses and purity of the peptides were analyzed using high-performance liquid chromatography as previously described (18). The synthesized peptides were prepared as a 10 mM stock solution in distilled water. For treatment, an aliquot of peptide stock solution was diluted RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin/streptomycin; both HyClone; Cytiva). The 3D structure of MP06 was predicted using PEP-FOLD3 (bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3) and PyMOL 3.0 software (pymol.org).

The human H1299 lung cancer cell and MRC5 lung normal fibroblast cell lines (Korean Cell Line Bank; cat. no. 25803 and 10171) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics. Human umbilical vein endothelial cells (HUVECs; American Type Cell Culture, CRL-1730) were incubated in endothelial basal medium-2 (EBM; cat. no. CC-3156) containing endothelial cell growth medium supplements (cat. no. CC-4176; both Lonza). Cells were cultured at 37°C in a humidified 5% CO₂ incubator. HUVECs were used between passages 3 and 4.

To investigate cytotoxicity, H1299 and human umbilical vein endothelial cells (HUVEC) were seeded at 5×10³ cells/well into a 96-well plate and treated with MP06 peptide at 5, 10 and 20 μM at 37°C for 24 h. Subsequently, the medium was replaced with 100 μl fresh RPMI-1640 and EBM containing 10 μl Cell Counting Kit-8 (Dojindo Molecular). After 3 h, the absorbance was measured using the Spectramax i3x (Molecular Devices) at 450 nm. To suppress AQP3 expression, H1299 cells were transfected with 10 pmol small interfering (si)RNA)-AQP3 (cat. no. sc-29713; Santa Cruz Biotechnology) and negative siRNA control (cat. no. sc-37007) by Lipofectamine RNAi MAX (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h according to the manufacturer's instructions. H1299 cells were cultured at 37°C in a humidified 5% CO₂ incubator for at least 48 h after transfection.

To evaluate the expression of genes, total RNA from H1299 cells and HUVECs was isolated by TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The purity and quality of RNA were determined using a UV spectrophotometer. First-strand cDNA was synthesized using the cDNA synthesis kit (iNtRON Biotechnology). PCR was performed using AccuPower PCR preMix, Bioneer, S. Korea). Amplification with specific primers (Table I) was conducted as follows: Initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 5 min, annealing at 56°C for 1 min and extension at 72°C for 1 min and final extension at 72°C for 3 min. The amplified PCR gene products were analyzed by 1% agarose gel electrophoresis containing redsafe (iNtRON Biotechnology) and imaged under UV light. β-actin was used as a reference gene.

H1299 and HUVEC cells were lysed by vortexing with RIPA lysis buffer containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich; Merck KGaA). The supernatant was centrifuged at 13,000 × g at 4°C for 30 min and protein concentration was measured using the Bradford assay (Bio-Rad Laboratories, Inc.). Equivalent amounts (30 μg) of protein were boiled for 5 min and separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking the membranes with a solution of tris-buffered saline (TBS) containing bovine serum albumin (BSA, 1%, Sigma-Aldrich; Merck KGaA; cat. no. A3294) at room temperature for 1 h and incubated overnight at 4°C with primary antibodies (all 1:1,000) against SOX2 (cat. no. sc-365823), Octamer binding transcription factor (OCT)3/4 (cat. no. sc-5279), Kruppel-like factor (KLF)4 (cat. no. sc-365144), CD44 (cat. no. sc-7297), N-cadherin (cat. no. sc-59987), E-cadherin (cat. no. sc-8426), Zinc-finger E-box-binding homeobox (ZEB)1 (cat. no. sc-515797), vimentin (cat. no. sc-6260), Snai1 (cat. no. sc-271977), AQP3 (cat. no. sc-518001) and β-actin (cat. no. sc-47778; all Santa Cruz Biotechnology) and VEGF (Bioswamp; cat. no. PAB30976). After 1 h incubation at room temperature with HRP-conjugated secondary antibodies (1:10,000; cat. nos. sc-2748 and rabbit sc-2357, Santa Cruz Biotechnology), membranes were rinsed with Tris-buffered saline and visualized using a western blotting substrate (Thermo Fisher Scientific, Inc.; cat. no. A38555).

H1299 cells (5×10⁴) were seeded on cover glass in cell culture plates. The cells were fixed using 4% paraformaldehyde for 30 min at room temperature, washed and blocked with phosphate-buffered saline (PBS) containing 1% BSA (Sigma-Aldrich; Merck KGaA) at room temperature for 40 min. Cells incubated with anti-SOX2 (cat. nosc-365823; 1:500) and anti-vimentin (cat. no. sc-6260; 1:500) in a solution of PBS at 4°C overnight. Then, the cover glass was washed with PBS and incubated with Alexa Fluor 488-conjugated antibody (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. A21202; 1:1,000) for 1 h at room temperature. Then cells were mounted with aqueous mounting containing DAPI at room temperature for 5 min (Vectashield Mounting Medium with DAPI H-1,200; Vector Laboratories). Cell images were acquired using a Zeiss LSM510 Meta fluorescence microscope at 40X magnification with ZEN 3.1 software. (Carl Zeiss GmbH).

Stem cell-permissive medium was prepared with DMEM-F12 (Cat. No. 11320-033; Invitrogen) supplemented with 20 ng/ml epidermal growth factor (E9644; Sigma-Aldrich), 20 ng/ml basic fibroblast growth factor (13256-029; Invitrogen) and B27 serum-free supplement (Gibco; Thermo Fisher Scientific, Inc). AggreWell 400(STEMCELL, #34415) or 800(STEMCELL, #34425) microwell plates were pretreated and washed with anti-adhesion solution(STEMCELL, #07010) for 5 min at 37°C. Then, H1299 cells were seeded at 1×10⁵ cells/well and centrifuged at 100 × g for 3 min at room temperature to capture cells inside the microwells with stem cell-permissive medium. Cells were incubated at 37°C with 5% CO₂ for 7-10 days. The formed H1299 spheroids were imaged using an inverted phase contrast microscope (Olympus Corporation; CKX53 light microscope; magnification, ×100).

For wound healing assay, H1299 (1×10⁵) cells were seeded on a 6-well plate. When cells reached 80% confluence, a wound was introduced across the diameter of each well using a 200-μl pipette tip. Images were captured by inverted phase contrast light microscopy, after 12 and 24 h in serum-free RPMI-1640 media with MP06 peptide. The healing area was quantified using ImageJ 1.54g software (National Institutes of Health).

The migration and invasion assay was conducted using a Transwell chamber (8-μm pores; BD Biosciences) in a 24-well plate. H1299 (2×10⁴) cells were seeded in the upper chambers with 200 μl serum-free RPMI-1640 medium with or without MP06 peptide at 37°C in a humidified 5% CO₂ incubator for 24 h. The lower chamber contained 500 μl RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin. For the invasion assay, a Transwell chamber was coated with diluted Matrigel (Corning, Inc.) for 30 min at 37°C. The migratory and invasive cells from the upper chamber were fixed with 4% paraformaldehyde for 20 min at 37°C and stained with crystal violet for 5 min at room temperature. The upper surface of Transwell membrane was wiped using a cotton swab to remove non-migratory and -invasive cells. Cells were then imaged using an inverted light microscope (Olympus CKX53; magnification, ×40).

HUVECs (1×10⁴) were seeded to 80% confluency for final passage at passage 2 into a 96-well plate. Each well was coated with 50 μl Geltrex matrix (Gibco; Thermo Fisher Scientific, Inc.) and allowed to solidify at 37°C for 30 min. H1299 culture media was collected, supernatant was centrifuged at 13,000 × g for 10 min at 4°C. Conditioned media (CM) were produced using mixed fresh EBM media/H1299 cultured media with MP06 or siAQP3 ratios (75:25, 50:50). The HUVECs were incubated at 37°C with 5% CO₂ for 24 h and stained with Calcein-AM (Invitrogen) at 37°C for 5 min. Angiogenesis was observed using an inverted light microscope at 100X magnification.

Zebrafish (Danio rerio) were provided by Professor C-H. Kim (Chungnam National University) and maintained as described in a previous study (31). Wild-type and transgenic (kdrl:eGFP) embryos were obtained by breeding males and females (2:2) in a 14/10-h light/dark cycle at 28.5°C with a recirculating water system. For anti-angiogenesis assay, fertilized zebrafish embryos were transferred to a 24-well plate (10 specimens/well) at the 70% epiboly stage. Embryos were exposed to 2 μM MP06 peptide by dissolving in egg water (60 μg/ml sea salt in distilled water). The zebrafish embryos were incubated for 24 h at 28.5°C. Embryos were anesthetized and mounted in 3% methylcellulose (Sigma-Aldrich; Merck KGaA) and then photographed using a fluorescence microscope at 40X magnification. (Leica DM6 B; Leica GmbH).

The AQP3 of expression profile of lung adenocarcinoma in tumor and normal tissue was analyzed from Gene Expression Profiling Interactive Analysis web server (gepia.cancer-pku.cn/detail. php?gene=AQP3, accessed April 24, 2024). This web server extracts data from TCGA data portal and Genotype-Tissue Expression (GTEx) database of normal tissue. Kaplan-Meier survival was analyzed using Gene Expression database of lung normal and tumor tissues 2 on expression of AQP3 (gent2.appex.kr/gent2/, accessed March 24, 2024).

Data are presented as the mean ± standard error of the mean. All experiments were conducted in triplicate. Comparisons were performed using two-tailed unpaired Student's t test or one-way ANOVA and Tukey's post hoc test for multiple comparisons. GraphPad Prism 10.3.1 (Dotmatics) was used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

MP06 tertiary structure was predicted using PEP-FOLD3 modeling (32). MP06 contained an N-terminal α-helix, unstructured C-terminal region and several basic amino acids that make its net charge positive (Fig. 1A and B). The charged amino acids were concentrated on one side of the helix, whereas hydrophobic amino acids formed the other side (Fig. 1C). FITC-MP06 was synthesized to examine its cellular effects and distribution (Fig. S1). FITC-MP06 accumulation was observed in the cytoplasm of H1299 cells (Fig. 1D). Morphological changes from spindle shape to a cobblestone-like shape were observed in cells treated with 20 μM MP06 or FITC-MP06 when compared to the untreated control cells (Fig. 1E). There was a dose-dependent decrease in viability in cells treated with MP06 or FITC-MP06, with a decrease in the cell viability of up to ~38% of the cells treated with MP06 (Fig. 1F). Collectively, these results indicated that MP06 and FITC-MP06 efficiently infiltrated cellular membranes, were localized in cytoplasm, and increased cytotoxicity in lung cancer cells.

CSC transcription factors (such as SOX2, OCT4, KLF4 and CD44) are important marker of cancer and normal stem cells (35,36). Following treatment with MP06, spheroid size significantly decreased compared with control H1299 cells (Fig. 2A). MP06 decreased expression of stemness markers (SOX2, OCT4, KLF4 and CD44), which indicated decreased self-renewal potential (Fig. 2B) Suppressed SOX2 expression was detected by immunofluorescence in FITC-MP06-treated H1299 cells (Fig. 2C). The results showed that MP06 partly regulated suppression of self-renewal activity in H1299 cells.

To investigate the metastatic function of MP06 on H1299 cells, EMT-associated properties were investigated by wound healing, migration and invasion assay. A significant decrease in the wound gap closure was observed after MP06 treatment (Fig. 3A). MP06-treated cells exhibited a significant decrease in migration and invasion abilities (Fig. 3B). MP06 notably decreased the cellular levels of important EMT markers (such as N-cadherin, ZEB1, vimentin and Snail) that regulate the migration of mesenchymal cells. E-cadherin, a representative marker of epithelial cells, expression increased (Fig. 3C). Reduced expression of vimentin was confirmed by immunofluorescence staining in FITC-MP06-treated H1299 cells (Fig. 3D). Collectively, these results suggested the involvement of MP06 in migration and invasion via decreased EMT marker expression in H1299 cells.

Effective angiogenesis is key for the growth of the primary tumor and the cancer cells' ability to spread to other locations in the body. To investigate the angiogenic ability of MP06, HUVECs, which can form tube-like structures depending on the VEGF content in the supernatant (38), were used. MP06-induced cytotoxicity in HUVECs was significantly lower than that in H1299 cells (Fig. 4A). MP06 notably decreased VEGF expression in H1299 cells (Fig. 4B). MP06-CM treatment decreased tube formation compared with that in the control H1299 culture media, implying that MP06 suppressed angiogenesis activity at a high rate with MP06-CM (Fig. 4C). The formation of tube-like structures and expression of VEGF in HUVECs was directly inhibited by MP06-treated EBM (Figs. 4D and S2). These results suggested the involvement of MP06 in VEGF-mediated tube formation and blockade of angiogenic responses.

MP06 can be crucial for angiogenesis potential of zebrafish embryos. A concentration of 2 μM MP06 was used to observe inhibition of angiogenesis, as concentrations >4 μM resulted in zebrafish embryo death. Changes in vascular patterning following MP06 treatment were observed during zebrafish development, particularly in transgenic zebrafish expressing the Tg (kdrl:eGFP) marker. MP06-treated embryos exhibited reduced growth of intersegment vessels (ISVs) at the apex compared with untreated control embryos in lateral view (Fig. 5A and B). In untreated control embryos, robust ISV growth was observed (filled arrowheads) compared with MP06-treated embryos, which showed noticeably decreased ISV growth (empty arrowheads) at the top of the embryo (Fig. S3). Quantification of the proportion of completed ISV structures in MP06-treated zebrafish at 30 h post-fertilization revealed a significant decreased of ~2.7-fold compared with that in untreated controls, indicating an adverse effect of MP06 treatment on vascular development (Fig. 5C and D). These angiogenic events, accompanied by decreased proliferation and migration in endothelial cell, support the role of MP06 as a negative regulator of angiogenesis.

Expression levels of AQP3 gene and protein were higher in H1299 cells than in normal cells (MRC5 cells and HUVECs; Fig. 6A). Furthermore, it was confirmed that AQP3 was more highly expressed in lung cancer than in normal lung tissue using Gene Expression Profiling Interactive Analysis web server (gepia.cancer-pku.cn/detail.php?gene=AQP3, accessed April 24, 2024; Fig. 6B). Gene expression profiling revealed that AQP3 was markedly upregulated in lung adenocarcinoma tissue. Additionally, the Kaplan-Meier survival analysis using Gene Expression database of normal and tumor tissues 2 (gent2.appex.kr/gent2/, accessed March 24, 2024) revealed an association between AQP3 and lung cancer. The Kaplan-Meier graph confirmed that the survival rate of patients with lung cancer and high AQP3 expression was low (Fig. 6C). H1299 cells treated with MP06 showed decreased levels of AQP3 expression (Fig. 6D). These results indicated that high AQP3 expression was associated with malignancy and decreased survival in lung cancer.

AQP3 gene was commonly overexpressed in H1299 cells compared with normal cells (Fig. 6A). AQP3 was knocked down to determine its effects on stemness, EMT and angiogenesis-associated changes in H1299 cells. Expression of the marker proteins SOX2, OCT4, KLF4 and CD44 was decreased following AQP3 knockdown, which was consistent with the decreased spheroid size (Fig. 7A and B). AQP3 knockdown decreased expression of EMT markers such as N-cadherin, ZEB1 and Snail, accompanied by decreased migration/invasion of H1299 cells (Fig. 7C and D). siAQP3-CM decreased tube formation compared with that in control H1299 culture medium, suggesting that AQP3 knockdown suppressed angiogenesis activity in siAQP3-CM-cultured cells (Fig. 7E). VEGF secretion regulates CSC and EMT phenomena through autocrine action (36). Consistent with VEGF expression in MP06-treated H1299 cells, VEGF expression consistently decreased in H1299 cells treated with siRNA-AQP3 (Fig. 7F).

Altogether, the in vitro and in vivo data suggested that MP06 was partially associated with cancer stemness and EMT in H1299 cells and that suppressing AQP3 expression to target VEGF may be an effective anti-angiogenic therapy.