XYA-2: a marine-derived compound targeting apoptosis and multiple signaling pathways in pancreatic cancer

Cell culture

PANC-1 and MIA-PaCa2 cell lines were obtained from the American Type Culture Collection. The cells were cultured in DMEM complete medium supplemented with 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY, USA) and 100 U/mL penicillin-streptomycin solution (Biosharp, Hefei, China). All cell cultures were maintained at a temperature of 37 °C in a 5% CO₂ incubator.

Acquisition of XYA-2

XYA-2 (N-(3,7-Dimethyl-2,6-octadienyl)-2-aza-2-deoxychaetoviridin A) was prepared by the same method as previously reported (Guan et al., 2023; Wang et al., 2020) and its purity was higher than 98%. Briefly, XYA-2 was extracted by extracting the fermentation broth, followed by purification by chromatography and preparative HPLC.

Cell viability assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. PANC-1 and MIA-PaCa2 cells were seeded at a density of 5 × 10³ cells per well in 96-well plates overnight. Different concentrations of XYA-2 included 1.56, 3.25, 6.25, 12.5, 25, 50, or 100 μM, were added for 24- and 72-h treatment periods. Following the treatment, 10 μL of CCK-8 reagent (Biosharp, Hefei, China) was added to each well, and the absorbance was measured at 450 nm after a 2-h incubation period with the Spark multimode microplate reader (TECAN, Grödig, Austria).

The principles guiding the selection of concentrations for the following in vitro experiments assessing the anti-tumor activity of XYA-2 are as follows: Integrating the goals of various experiments with considerations for cell density, drug exposure duration, and established IC₅₀ values, we opted to assess a range of concentrations, including low, medium, and high. This approach aims to establish a dose-response relationship and comprehend the XYA-2’s impact on tumor cells across a spectrum of concentrations. If high concentrations result in extensive cell death, a reduction in concentration is considered to facilitate the evaluation of experimental parameters.

Colony formation assay

Data were collected following established procedures (Yu, 2022 #43). Briefly, PANC-1 and MIA-PaCa2 cells were seeded in six-well plates at a density of 2,000 cells per well. Following adherence, cells were treated with either 0 or 2 µM XYA-2 for 24 h. Subsequently, the medium was replaced, and cells were cultured at 37 °C for 10–14 days. Colonies were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. Quantification of visible colonies was conducted using ImageJ software.

Cell cycle arrest assay

PANC-1 and MIA-PaCa2 cells were seeded at 2 × 10⁶ cells per dish. per 10-cm dish overnight and treated with either DMSO (control) or XYA-2 at 2 or 5 μM for 24 h. Then the cells were trypsinized, centrifuged at 1,000 rpm for 5 min at room temperature, and washed three times with 1 mL pre-chilled 75% ethanol. Subsequently, the cells were fixed overnight at −20 °C. Following fixation, the cells were re-suspended and stained with 500 µL PI/RNase staining buffer at room temperature for 30 min. The Attune NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA) was employed to detect the stained cells, and the results were analyzed using FlowJo software.

Apoptosis assay

PANC-1 and MIA-PaCa2 cells were seeded at 1 × 10⁶ cells per 6-cm dish overnight and treated with either DMSO (control) or XYA-2 at 5 or 10 μM for 24 h. Then the cells were re-suspended in 500 μL of 1X binding buffer. Subsequently, 5 μL of fluorescein isothiocyanate-coupled annexin and 5 μL of propidium iodide were sequentially added to the cell suspension and incubated for 15 min in the dark at room temperature. The Attune NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the fraction of apoptotic cells. The data analysis categorized the results as follows: Q1 (top left): necrotic cells, Q2 (upper right): late apoptotic cells, Q3 (bottom right): early apoptosis, and Q4 (lower left): cells that did not undergo apoptosis. The apoptosis rate was calculated as the sum of the percentages from Q2 and Q3 quadrants.

Western blot assay

Human pancreatic cancer cell lines PANC-1 and MIA-PaCa2 were cultured to 40–50% confluency. Treatment with the compound XYA-2 was performed at concentrations of 0, 5, 10, or 20 μM for 24 h. Following treatment, cells were washed with phosphate-buffered saline and lysed using RIPA buffer containing protease inhibitors. The resulting cell lysates were centrifuged, and the supernatants were collected to obtain total protein. Western blot analysis was carried out by separating equal amounts of protein using SDS-PAGE, followed by transfer to a polyvinylidene fluoride membrane. The membrane was then blocked and incubated with specific antibodies targeting Caspase 3, Cleaved Caspase 3, PARP, Cleaved PARP, Stat3, and Phospho-Stat3 (Tyr705). Immunoreactivity was detected using chemiluminescence, and Western blot results were quantified using an image analysis system. The antibodies used in this study were as follows: Caspase 3: Cell Signaling Technology (CST), #9662f; Cleaved Caspase 3: Proteintech, #19677-1-AP; PARP: CST, #9532; Cleaved-PARP: CST, #5625; Stat3: CST, #12640; Phospho-Stat3 (Tyr705): CST, #9145.

Wound healing assay

Upon reaching 80–90% confluency, PANC-1 and MIA-PaCa2 cells were subjected to scratch wound assays. Wounds were generated by scraping the cell monolayers, followed by treatment with various concentrations of XYA-2 compound (0, 2, 5, or 10 μM). The cells were observed using the CKX53 Inverted Microscope (Olympus, Tokyo, Japan) and imaged at 0, 24, and 36 h after scratching to evaluate cell migration at the edges of the scratch area.

Transwell migration assay

A total of 2 × 10⁵ cells in 200 μL of serum-free media, along with 0, 2, or 5 μM XYA-2 were seeded into the upper chamber. The lower chamber was filled with 600 μL of DMEM containing 10% FBS. After incubating at 37°C for 48 h, the cells on the upper membrane were removed using cotton swabs. The cells that had migrated to the lower membrane were fixed with 4% formaldehyde and stained with 2.5% crystal purple. Subsequently, the invaded cells were counted in five different fields under the CKX53 Inverted Microscope (Olympus, Grödig, Japan). The stained cells were observed and photographed using an inverted microscope. Cells that had migrated to the underside of the gel layer appeared as stained spots at the bottom of the chamber. To quantify the extent of migration, the stained spots could be counted using ImageJ software.

RNA-sequencing and bioinformatics analysis

The RNA-seq samples was prepared as previously described (Guan, 2023 #42). Specifically, PANC-1 and MIA-PaCa2 cells were seeded in 10 cm culture dishes at a density of 2 × 10⁶ cells per dish. Experimental groups, comprising a control treated with DMSO and another treated with 10 µM XYA-2 for 24 h, each included three biological replicates. Post-incubation, RNA extraction was performed using Trizol, and RNA-seq data generation was conducted by LC-Bio Technology Co., Ltd. (Hangzhou, China). For the construction of the RNA-seq transcriptome library, 1 µg of total RNA from each of the three biological replicates was utilized, employing the TruSeq TM RNA sample preparation Kit from Illumina (San Diego, CA, USA). The acquired data were subsequently meticulously analyzed using the free online platform provided by Lianchuan Cloud Platform. Differential expression analysis was performed using the DESeq package in the R platform, utilizing the default configuration. Furthermore, gene set enrichment analysis (GSEA) was carried out using hallmark gene sets and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets from MSigDB.

RNA extraction, cDNA synthesis, and quantitative real-time PCR

Pancreatic cancer cells were treated with different concentrations of XYA-2 for 24 h, with DMSO treatment used as a control. Each group consisted of three biological replicates. Total RNA was extracted using the protocol provided by the manufacturer of a commercially available RNA extraction kit (YiShan Biotechnology Co. LTD, Shanghai, China). DNase I treatment (RNeasy MinElute Cleanup Kit; QIAGEN, Hilden, Germany) was used to remove and assess the DNA contamination from RNA samples. The quality control of RNA was conducted by a NanoDrop spectrophotometer (ND-2000; Thermo Fisher Scientific, Waltham, MA, USA). The values of A260/A280 of all RNA samples were between 1.8 and 2.0. The Agilent 2100 Bioanalyzer instrument was used to calculate the RNA integrity number (RIN). Inhibition of reverse-transcription activity was checked by dilution of the sample.

For cDNA synthesis, 2 μg of total RNA was reverse-transcribed into complementary DNA (cDNA) using a reverse transcription kit (YiShan Biotechnology Co. LTD, Shanghai, China). The cDNA synthesis reaction was carried out at 37 °C for 60 min, followed by thermal inactivation at 95 °C for 5 min. The synthesized cDNA samples were then subjected to real-time quantitative polymerase chain reaction (qRT-PCR) using specific primers designed for the target genes. The primers used in this study are listed in Table 1. The total reaction volume was 20 μL, consisting of 10 μL SYBR Green Master Mix (QP002; ESscience Biotech Co. LTD, Shanghai, China), 0.4 μL forward primer, 0.4 μL reverse primer, 2 μL cDNA template, and ddH₂O added to reach a final volume of 20 μL. The final primer concentration was 200 nM.

qPCR was conducted as previously described (Qi, 2022 #44) utilizing the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). In brief, the reaction conditions comprised an initial pre-denaturation at 95 °C for 5 min, succeeded by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. Melt curve analysis was carried out to confirm amplicon specificity. The amplification products had lengths ranging from 110 to 300 bp. To ensure accurate normalization, GAPDH was used as the housekeeping gene and normalizer. All experiments were performed with three technical replicates. The relative gene expression levels were determined using the 2^−ΔΔCt method, with PCR efficiencies > 95% and the R² was > 0.990. The linear dynamic range, Cq variation at the lower limit, and confidence intervals throughout the range were assessed. Limit of detection for each assay were evaluated. Additionally, the qPCR analysis program used, including its source and version, was specified. The Cq method was employed for determination, and outlier identification and disposition methods were described. Results of negative template controls (NTCs) were reported. Repeatability (intra-assay variation) and reproducibility (inter-assay variation measured as %CV) were analyzed. Power analysis was conducted to determine the sample size.

Statistical analysis

The statistical analysis in this study is performed using GraphPad Prism 9 for macOS (Version 9.4.1; GraphPad Software, La Jolla, CA, USA). The data analysis was expressed as mean ± standard error of the mean (SEM). Student’s t-test was used to determine the significant differences between experimental groups and control groups. Statistical significance was considered if P < 0.05.

XYA-2 inhibits the proliferation of pancreatic cancer cells

To investigate the impact of XYA-2 on pancreatic cancer cells, a CCK-8 assay was initially conducted. The results from the CCK-8 assay revealed that XYA-2 effectively suppressed the activity of pancreatic cancer cells after 24 and 72 h of treatment (Figs. 1B and 1C). Moreover, the IC₅₀ values at 24 h were determined to be 13.62, 21.84, and 16.02 μM for MIA-PACA2, PANC-1, and PANC0203, respectively. At 48 h, these values were 3.148, 8.61, and 8.174 μM, and at 72 h, the IC50 values were found to be 1.141, 1.84, and 3.802 μM for MIA-PaCa2, PANC-1, and PANC0203, respectively. These findings suggest that XYA-2 exhibits concentration-dependent inhibition of pancreatic cancer cell activity. In light of these observed results, we have made the strategic decision to prioritize MIA-PaCa2 and PANC-1 for subsequent downstream experiments.

Furthermore, a colony formation assay was performed to assess the effect of XYA-2. We chose a concentration of 2 μM for the clonogenic formation experiment, which was slightly higher than IC₅₀ for 72 h to ensure a robust assessment of the inhibitory effects. The results of the assay showed that XYA-2 had a notable inhibitory effect on colony formation in pancreatic cancer cells. Specifically, in MIA PaCa-2 cells, the inhibition of colony formation was 33%, while in PANC-1 cells, it was more pronounced at 73% (Fig. 1C). These findings indicate that XYA-2 significantly hampers the colony formation ability of pancreatic cancer cells, underscoring its potential as a therapeutic agent for pancreatic cancer treatment.

XYA-2 induces cell cycle arrest and apoptosis in pancreatic cancer cells

XYA-2 exerts an influence on the cell cycle of pancreatic cancer cells. As illustrated in Fig. 2A, treatment with 2 μM XYA-2 led to a significant increase in the number of PANC-1 cells in the G2/M phase, while concurrently reducing the number of cells in the G0/G1 phase. These results indicate that XYA-2 can effectively impede the transition of PANC-1 cells from the G2 phase to the M phase, thereby inhibiting cell proliferation.

XYA-2 also promotes apoptosis in pancreatic cancer cells. At a concentration of 5 μM, the apoptosis rate was measured as 5.48% for MIA-PaCa2 and 9.18% for PANC-1 cells. When the concentration was doubled, the apoptosis rates increased to 9.60% for MIA-PaCa2 and 12.90% for PANC-1 cells (Figs. 2B and 2C). We conducted Western blot analysis targeting apoptosis-related markers, including caspase-3, cleaved caspase-3, PARP and cleaved PARP. Our results reveal that, as the concentrations of XYA-2 increased, the protein levels of caspase-3 and PARP decreased, while the levels of cleaved PARP increased. Notably, there was no concurrent increase in cleaved caspase-3 (Fig. 2D). We considered the possibility of other caspases, such as caspase-7, exhibiting functional similarities and sharing PARP as a substrate. The observed rise in cleaved PARP provides evidence of an overall increase in apoptosis. However, the precise mechanisms governing PARP cleavage still necessitate further exploration. Furthermore, our analysis extended to STAT3 and phosphorylated STAT3, recognized targets of XYA-2 in its anti-gastric cancer activity. Our results demonstrate that XYA-2 inhibits STAT3 phosphorylation in pancreatic cancer cells (Fig. 2D). This suggests that XYA-2 may, at least in part, exert its anti-cancer effects through the modulation of apoptosis-related proteins and the inhibition of STAT3 phosphorylation in pancreatic cancer cells.

XYA-2 exhibits concentration-dependent inhibition of invasion and migration in pancreatic cancer cells

The results from the wound healing assay, depicted in Fig. 3A, underscore the concentration-dependent inhibitory effects of XYA-2 on the migratory capacity of MIA-PACA2 cells. After 24 h, the migration rates for MIA-PACA2 cells treated with 2, 5, and 10 μM XYA-2 were 22.3%, 8.6%, and 6.4%, respectively. Subsequently, at 36 h, the migration rates for MIA-PaCa2 cells exposed to 2, 5, and 10 μM XYA-2 were recorded as 41.9%, 22%, and 4.1%, respectively. Figure 3B presents the transwell assay results, highlighting XYA-2’s inhibitory effects on the invasive capability of PANC-1 and MIA-PaCa2 cells. Notably, reductions of 60% and 36% were observed in PANC-1 and MIA-PaCa2, respectively, at a concentration of 2 μM XYA-2. Importantly, these inhibitory effects on invasive ability surged significantly, reaching 99.2% for PANC-1 and 64% for MIA-PaCa2 when treated with 5 μM XYA-2. These findings affirm the dose-dependent impact of XYA-2 on the migration and invasion of pancreatic cancer cells.

XYA-2 as a potential inhibitor of pancreatic cancer cell progression through apoptosis induction and immune modulation

To elucidate the mechanism underlying the inhibitory effect of XYA-2 on pancreatic cancer cell progression, transcriptome analysis was conducted on cells treated with 10 μM XYA-2 and compared to control cells. In MIA-PaCa2 cells, 3,745 genes were found to be upregulated and 2,089 genes were downregulated in XYA-2-treated cells compared to the control group. Similarly, in PANC-1 cells, both upregulated and downregulated genes in XYA-2-treated cells were identified, totaling 4,324 genes. To account for any ontogenetic differences between the two cell strains, the intersection of upregulated and downregulated genes was identified separately. Ultimately, 1,933 upregulated genes and 1,158 downregulated genes were identified in XYA-2-treated cells (Fig. 4A).

Following this, GSEA enrichment analysis was conducted on the dysregulated genes. Examination of hallmark gene sets revealed predominant enrichment of proliferation-related gene sets, such as “G2M checkpoint,” “E2F targets,” and “mitotic spindle,” in the DMSO treatment group. In contrast, the gene set “apoptosis” was significantly enriched in the XYA-2 treatment group (Fig. 4B). This suggests a pivotal role of XYA-2 in inducing apoptosis and impeding cell proliferation. Additionally, analysis of KEGG gene sets indicated prominent enrichment of immune-related pathways, specifically “antigen processing and presentation” and “natural killer cell-mediated cytotoxicity,” in the XYA-2 treatment group (Fig. 4B). This implies potential implications of XYA-2 in modulating immune responses against pancreatic cancer cells.

XYA-2 downregulates critical genes, associated with better survival in pancreatic cancer, and shows potential as a regulator of pancreatic cancer progression

Based on the results of the transcriptome analysis described above, we screened the top ten downregulated genes, namely ESPL1 (extra spindle pole bodies like 1, separase), PIF1 (PIF1 5′-to-3′ DNA helicase), PLK1 (polo like kinase 1), SPC24 (SPC24 component of NDC80 kinetochore complex), CCNF (cyclin F), TUBGCP5 (tubulin gamma complex component 5), KIF2C (kinesin family member 2C), KIF4A (kinesin family member 4A), TOP2A (DNA topoisomerase II alpha), and MKI67 (marker of proliferation Ki-67) (Fig. 5A). Further analysis using the public database GEPIA revealed that the expression of six genes (PLK1, SPC24, KIF2C, TOP2A, MKI67, and KIF4A) was significantly associated with overall survival in pancreatic cancer patients. Notably, low expression levels of these genes were strongly correlated with higher survival rates in pancreatic cancer patients (P-values ranging from 0.0019 to 0.036) (Fig. 5B). To validate these findings, we analyzed the expression of these genes in clinical samples of pancreatic cancer. As shown in Fig. 5B, the expression levels of these genes were significantly higher in pancreatic cancer tissue compared to adjacent normal tissue. Furthermore, existing reports suggest that these genes are involved in the development and progression of various types of cancer (Uusküla-Reimand & Wilson, 2022; Wei et al., 2021; Wu et al., 2021; Zhu et al., 2015). To further investigate the influence of XYA-2 on the expression of key genes and its role in pancreatic cancer cell development, we treated MIA-PaCa2 cells and PANC-1 cells with different concentrations of XYA-2. Subsequent qRT-PCR analysis revealed a significant downregulation of all tested genes in XYA-2-treated cells, regardless of whether the concentration used was 5 or 10 μM (Fig. 5C). These results further support the notion that XYA-2 may play a crucial role in regulating the expression of key genes involved in pancreatic cancer progression.