A rearranged abietane diterpenoid from Clerodendrum mandarinorum inhibits tumor progression of oral squamous cell carcinoma in vitro

2.1 General experimental procedures

General experimental procedures for isolation and structure elucidation were included in the Supporting Information.

2.2 Plant material

The twigs and leaves of Clerodendrum mandarinorum Diels were collected from Wutai Mountain, Yunnan province, China in October 2020 and identified by one of authors (C. X.). The voucher specimen (No. 202010CM) was deposited at the College of Pharmacy, Nankai University, China.

2.3 Cell lines and cell culture

Human nasopharyngeal carcinoma cells CNE, human oral squamous cell carcinoma cells CAL-27, non-small cell lung cancer cells A549, and pancreatic cancer cells PANC-1 were cultured with RPMI-1640 medium that contained 10 % fetal bovine serum and grown under standard conditions (37 °C, 95 % air atmosphere, 5 % CO₂).

2.4 Cell viability assay

Cells were seeded in 96-well plates at a density of 3000 cells/well, and cultured in a cell culture incubator at 37 °C and 5 % CO₂ for 24 h. Drugs with concentrations of 0 μM, 15 μM, and 30 μM were prepared in their respective culture media and added to the wells of the 96-well plate for a 48-hour incubation period. MTT (Thiazolyl Blue) solution (5 mg/ml) was co-incubated with the cells at 37 °C and 5 % CO₂ for 4 h to assess cells viability. The blue-purple crystals formed in the 96-well plate were completely dissolved using Dimethyl sulfoxide (DMSO), and the absorbance (OD value) was measured at a wavelength of 570 nm by a microplate reader. The obtained data was analyzed by GraphPad Prism 9.0 (Stockert et al., 2018).

2.5 EDU (5-ethynyl-2-deoxyuridine) cell proliferation assay

The experiment involved treating CAL-27 cells with various concentrations of compound 1 for 24 h. After the treatment period, the cells were incubated with EDU working solution for 2 h. This solution specifically labels newly proliferated cell DNA molecules. Following the incubation with the EDU working solution, the cells were fixed and permeabilized to prepare them for further analysis. A click reaction solution was added to the cells at room temperature, and then the cells were incubated in darkness for a duration of 30 min. This click reaction solution reacts with the EDU-labeled DNA molecules, allowing for their easy detection and visualization. Finally, the labeling status of the cells was determined using a specialized instrument called a confocal microscope (Xiao et al., 2023).

2.6 Wound healing assay

To initiate the experiment, prepare a 24 well plate with a specific cell density of 3 × 10⁵ cells per well. Gently glide a sterilized pipette tip across the well surface, ensuring consistent size and depth of the scratch gaps created. Once the scratch gaps are formed, cautiously add RPMI-1640 medium to each well, ensuring that the medium contains different concentrations of the drugs under investigation. Capture images of the scratch gaps at 0, 12, 24, and 36 h post-treatment. Software tools such as Image J and GraphPad Prism9.0 were employed for analysis and data processing (Lin et al., 2022).

2.7 Transwell assay

Mix matrigel and RPMI-1640 medium in a ratio of 1:3. Then add this mixture to the transwell chamber and let it to stand for 30 min at a temperature of 37 °C. Before seeding the cells, it is important to hydrate the basal membrane. This can be achieved by adding a culture medium containing 0.1 % fetal bovine serum to the transwell chamber and incubating it for 30 min. Next, collect the desired cells and resuspend them in a culture medium containing 0.1 % fetal bovine serum. 2 × 10⁵ cells were added to each transwell chamber. To induce cell invasion, add a complete culture medium to the lower layer of the transwell chamber. Place the transwell chamber in a cell culture incubator set to a temperature of 37 °C and a CO₂ concentration of 5 %. Allow the cells to invade for 24 h. Remove any remaining cells from the upper side of the transwell chamber by gently wiping it with a cotton swab. After removing the residual cells, fix the cells that have invaded the lower side of the transwell chamber by adding 4 % cell fixative to the chamber and incubating it for 30 min. Finally, stain the cells with crystal violet dye. After staining, the cells can be observed and captured under a microscope for further analysis (Xiao et al., 2023).

2.8 Colony formation experiment

The cells were digested with pancreatic enzyme and collected by centrifugation. Subsequently, the cells were seeded into 6-well plates at a density of 300 cells per well. After 24 h of incubation, different concentrations of compound 1 were added to the wells. The plates were then placed in a cell culture incubator set at 37 °C and 5 %CO₂ for a period of 7 days. After incubation, the cells were fixed with 4 % cell fixative solution for 30 min. Following fixation, the cells were stained with a crystal violet solution. To quantify the number of cell clones, statistical analysis was performed using the Image J (Xiao et al., 2023).

2.9 Cellular thermal shift assay

The cells were initially treated with either PBS or compound 1 (30 μM) for 24 h. After this treatment, the cells were digested using pancreatic enzymes and collected by centrifugation. The cells suspended in PBS underwent another round of centrifugation. To prepare the samples for further analysis, the cells were re-suspended in RIPA lysate, which contained equal amounts of either PBS or compound 1. The cell suspension was divided into separate EP tubes, with each tube containing 80 μL of the cell suspension. Next, the cells in each tube were exposed to different temperatures ranging from 45 °C to 75 °C for a duration of 5 min each. Following the heating process, the cells were rapidly frozen in liquid nitrogen. This freezing and thawing process was repeated three times. After the freezing-thawing cycles, the cell lysate obtained from the samples was centrifuged at a rotating speed of 20,000×g for 20 min at a temperature of 4 °C. The supernatant was collected for further analysis using western blot (Jafari et al., 2014).

2.10 Western blotting

CAL-27 cells were treated with different concentrations of compound 1 (0 μM, 15 μM and 30 μM) for 24 h. After the treatment, RIPA lysate was added to the cells to collect the total protein. The protein concentration was measured using the BCA reagent. To further analyze the protein samples, the same samples were separated using a 10 % gel and transferred to a PVDF membrane. The PVDF membrane was sealed with 5 % skim milk for 1 h to ensure proper binding. Subsequently, the primary antibody was incubated with the membrane overnight at 4 °C. The second antibody was added to the membrane and incubated at room temperature for 2 h. The protein expression bands corresponding to the target proteins were detected using a chemiluminescence detector. The resulting images were quantitatively analyzed by Image J (Lin et al., 2022).

2.11 qRT-PCR

In this experiment, total RNA was extracted using the Trizol reagent. After extraction, the concentration of the RNA was measured using a microspectrophotometer, which enables precise quantification of nucleic acids by measuring their absorbance. The Fastking gDNA Dispelling RT Super Mix was used to perform reverse transcription synthesis of cDNA. This kit contains all the necessary components, including enzymes and primers, to effective conversion of RNA into complementary DNA (cDNA). For the subsequent quantitative polymerase chain reaction (qPCR), we used the Unicon qPCR SYBR Green Master Mix. This mix contains a fluorescent dye, SYBR Green, which binds to the newly synthesized DNA during the amplification process. The abundance of the target gene in the sample was measured by monitoring the increase in fluorescence (Xiao et al., 2023).

2.12 Immunofluorescence

CAL-27 cells were cultured on a cell creep in a 24-well plate for 24 h. Afterward, they were treated with various concentrations of compound 1 for an additional 24 h. Following this, the cells were fixed using a 4 % cell fixative solution for 30 min. To ensure effective permeability of the cell membrane, 0.2 % Triton X-100 was used. Additionally, to block any non-specific binding of proteins, 5 % BSA was applied. The primary antibody was then incubated overnight at a temperature of 4 °C, and the secondary antibody was incubated at room temperature for 2 h. The cell nuclei were stained using DAPI, and finally, the coverslip was sealed. The staining was subsequently observed using a confocal microscope (Xiao et al., 2023).

2.13 Statistical analysis

All experiments were performed in triplicates at least, all statistical analyses were performed using GraphPad Prism 8.0. The data are expressed as mean ± standard deviation (SD). The t-test was performed to compare the significant difference of the means between the two groups. All results were quantified using ImageJ software (NIH, Bethesda, MD, USA). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, and ^####p < 0.0001 were considered statistically significant.

3.1 Structural identification of compounds 1–6

The EtOAc-soluble layer from the methanol extract of the twigs and leaves of C. mandarinorum afforded six known rearranged abietane diterpenoids (Fig. 1), which were identified as 11-methoxyl-12,14-dihydroxy-13-(2-hydroxypropyl)-3,5,8,11,13-abietapentaen-7-one (1) (Xu et al., 2010), uncinatone (7,11-dihydroxy-3,4,9,11b-tetramethyl-1,2,8,9,11b-pentahydrophenanthro[3,2-b]furan-6(2H)-one, 2) (Tian et al., 1993), teuvincenone G [(16S)-12,16-epoxy-11,14-dihydroxy-17(15 → 16)-abeo-abieta-8,11,13-triene-3,7-dione, 3] (Cuadrado et al., 1992), teuvincenone H (12,16-epoxy-6,11,14-trihydroxy-17(15 → 16)-abeo-abieta-5,8,11,13,15-pentaene-3,7-dione, 4) (Pinheiro Barros et al., 2003), teuvincenone E [(16S)-12,16-epoxy-11,14-dihydroxy-17(15 → 16),18(4 → 3)-diabeo-abieta-3,5,8,11,13-pentaene-2,7-dione, 5] (Rodríguez, 2002), and villosin B [(5S,10S,16R)-12,16-epoxy-11,14,17-trihydroxy17(15 → 16)-abeo-abieta-8,11,13-triene-7-one, 6] (Li et al., 2014) by comparison of their spectroscopic data with those previously reported in the literature (Supporting Information). For compound 1, the ¹H NMR spectrum exhibited the presence of one methoxy group at δ_H 3.80 (s, 3H), four methyl groups at δ_H 1.15 (d, J = 6.2 Hz, 3H), 1.52 (s, 3H), 1.91 (s, 3H), 1.94 (s, 3H), one olefinic methine at δ_H 6.22 (s, 1H), and one oxygenated methine at δ_H 4.13 (s, 1H). The ¹³C NMR data revealed the presence of 21 carbon resonances, including one methoxy group at δ_C 61.3, one ketone at δ_C 192.2, one oxymethine at δ_C 68.1 and ten olefinic carbon resonances at δ_C 168.8, 156.4, 154.9, 143.1, 141.6, 136.1, 126.3, 119.6, 119.0, and 112.1. The absolute configurations at C-10 and C-16 were demonstrated as 10S and 16R according to ECD curve (Fig. S3, Murata et al., 2016; Fan et al., 2000) and comparison of the key ¹³C NMR data with the known analogue szemaoenoid C, whose absolute configuration was established by X-ray diffraction analysis (Table S1, Pu et al., 2018). The ¹³C NMR data of compound 3 was also provided for the first time in the Table S2 based on the 2D-NMR spectra.

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Fig. 1

Chemical structures of compounds 1–6.

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3.2 Compound 1 inhibits the proliferation, migration and invasion of oral squamous cell carcinoma in vitro

The effectiveness of all compounds except compound 3 and compound 5 (due to limited amount available) in inhibiting the growth of four types of human tumor cells, including CNE, CAL-27, A549 and PANC-1, was tested in vitro. The results showed that compound 1 exhibited the highest level of inhibition on CAL-27. At a concentration of 65.63 μM, half of the inhibitory effect on CAL-27 was achieved (Fig. 2A and B). Moreover, to further investigate the impact of compound 1 on CAL-27 proliferation, MTT assay and EdU cell proliferation assay were conducted, both of which demonstrated a dose-dependent inhibition patter (Fig. 2C and D). These assays provided evidence that compound 1 suppressed the proliferation of CAL-27. Additionally, the migration behavior of CAL-27 was examined using a wound healing assay. The results indicated that compound 1 significantly hindered the migration of CAL-27, with the level of inhibition increasing as the concentration of compound 1 increased (Fig. 2E). Furthermore, the result of transwell assay confirmed that compound 1 also effectively inhibited the invasive behavior of CAL-27. Similar to the pattern observed for proliferation inhibition, this inhibitory effect was also found to be dose-dependent (Fig. 2F). Lastly, a colony formation experiment was conducted to assess the impact of compound 1 on the colony formation ability of CAL-27. The results revealed that compound 1 significantly suppressed the ability of CAL-27 to form colonies (Fig. 2G). Overall, these findings demonstrate that compound 1 derived from C. mandarinorum possesses potent antitumor properties, particularly in inhibiting the growth, proliferation, migration, invasion and colony formation abilities of CAL-27, highlighting its potential as a promising therapeutic candidate for tongue squamous cell carcinoma treatment.

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Fig. 2

Compound 1 inhibited the proliferation, migration and invasion of CAL-27 in vitro. (A) Through MTT assay, the inhibitory rates of compounds 1, 2, 4, and 6 on four types of tumor cells were detected. (B) The half inhibitory concentration of the compound 1 on CAL-27 cells was tested by MTT assay. (C) MTT assay was used to determine the proliferation inhibition rate of compound 1 on Cal27 cells at concentrations of 0 μM, 15 μM, and 30 μM. (D) Expose CAL-27 to compound 1 at concentrations of 0 μM, 15 μM, and 30 μM for 24 h, then tested EDU fluorescence intensity. (E) Compound 1 inhibited wound healing of CAL-27 in a concentration-dependent manner. (F) Compound 1 inhibited the invasive ability of CAL-27 in a concentration-dependent manner. (G) Compound 1 inhibited the clonogenicity of CAL-27 in a concentration-dependent manner. ****p < 0.0001.

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3.3 Compound 1 inhibits the activation of the TGF-β/Smad and PI3K/AKT pathways, as well as epithelial-mesenchymal transition (EMT), through specific binding to TGFβR2

To further investigate the mechanism by which compound 1 inhibited tumor cell proliferation and metastasis, the PharmMapper database was used to predict its potential binding targets. It was discovered that compound 1 had a strong likelihood of binding to TGFβR2. This finding is particularly significant considering previous studies linking TGFβR2 to tumor development and metastasis (Xie et al., 2022). To authenticate the interaction between compound 1 and TGFβR2, molecular docking was employed. The results revealed that compound 1 bound to three specific amino acid residues (LYS277, ALA326, and ASP397) within the TGFβR2 structure (PDB ID 5E8V). Compound 1 occupied the protein kinase domain of TGFβR2 by interacting with the three amino acid residues, thereby inhibiting the formation of heterodimers and the activation of downstream signaling pathways. Furthermore, computer simulations were conducted to calculate the binding energy between compound 1 and TGFβR2, confirming their strong affinity (Fig. 3A). To further verify the binding between compound 1 and TGFβR2, a cellular thermal shift assay was performed. By subjecting the protein to increasing temperatures, a pronounced decrease in the protein degradation rate of TGFβR2 was observed in the presence of compound 1 compared to the control group treated with PBS (Fig. 3B). This provides compelling evidence for the binding interaction between compound 1 and TGFβR2 and supports the notion that compound 1 may play a role in inhibiting tumor cell proliferation and metastasis through its interaction with TGFβR2.

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Fig. 3

Compound 1 targets TGFβR2 in vitro, inhibiting the PI3K/AKT and TGF-β/Smad pathways as well as the EMT process. (A) Virtual molecule docking of compound 1 with TGFβR2 and binding energy between the two. (B) The binding ability of compound 1 to TGFβR2 was verified by cell heat transfer assay (C) The inhibition of TGF-β/Smad pathway activation by compound 1 was confirmed by Western blot (D) The inhibition of PI3K/AKT pathway activation by compound 1 was confirmed by Western blot (E) The protein expression levels of EMT process markers were detected by western blot. (F) The mRNA expression levels of EMT process markers were detected by qRT-PCR. (G) The protein expression levels of EMT process markers were detected by immunofluorescence. ^nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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TGFβR2 plays a crucial role in the TGF-β signaling pathway, which is involved in various cellular processes. In addition to the classical TGF-β/Smad pathway, TGFβR2 also activates non-classical pathways, including Wnt/β-catenin, PI3K/AKT, and NF-κB (Loomans and Andl, 2014). Previous studies have established the significance of the TGF-β/Smad pathway in tumor metastasis and progression, as well as the role of the PI3K/AKT pathway in non-classical signaling. To investigate the effects of compound 1 on these pathways, western blot analysis was performed to measure the phosphorylation levels of key proteins involved. The results demonstrated that compound 1 significantly decreased the phosphorylation levels of Smad2, Smad3, PI3K, and AKT (Fig. 3C and D). This indicated that compound 1 suppressed the activation of both the classical and non-classical TGF-β signaling pathways. Furthermore, compound 1 also influenced the expression of epithelial-mesenchymal transition (EMT) markers, which were regulated by the TGF-β signaling pathway. Immunoblotting analysis showed a reduction in the expression levels of Twist1, vimentin, and N-cadherin, while the expression of E-cadherin increased (Fig. 3E). Similarly, mRNA expression levels of vimentin and N-cadherin decreased, while the mRNA levels of E-cadherin increased (Fig. 3F). These findings suggest that compound 1 inhibits the occurrence of EMT, a crucial process for tumor metastasis. To further confirm these results, immunofluorescence staining was performed, which supported previous findings (Fig. 3G). Taken together, these results provide strong evidence that compound 1 effectively hinders the progression of EMT, thereby potentially suppressing tumor metastasis.

3.4 Transcriptome sequencing confirmed the inhibitory effect of compound 1 on TGF-β/smad and PI3K/AKT pathways

Furthermore, to gain a deeper understanding of the impact of compound 1 on CAL-27, RNA sequencing was utilized to analyze two distinct groups of cells: one treated with compound 1 and the other without treatment. The results depicted on the heat maps clearly showed notable differences in gene expression between these two groups (Fig. 4A). Additionally, the volcano plot provided intriguing insights, revealing that a total of 1556 genes were up-regulated, while 2438 genes were down-regulated in the cells treated with compound 1 compared to the untreated cells (Fig. 4B). To explore the biological significance of these differentially expressed genes, GO and KEGG cluster analysis was conducted. This analysis revealed that these genes were primarily associated with crucial processes such as protein biosynthesis and degradation, as well as significant diseases such as type II diabetes and human papillomavirus infection. Moreover, the enriched pathways obtained from the analysis included the TGF-β signaling pathway and the PI3K/AKT signaling pathway (Fig. 4C and D). These results are consistent with the in vitro research results mentioned above.

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Fig. 4

Transcriptome analysis. (A)Heat maps show the differential genes of CAL-27 cells treated with compound 1 for 24 h versus untreated cells. (B) The volcano map showed the number of up-regulated and down-regulated genes in CAL-27 cells treated with compound 1 for 24 h and in untreated CAL-27 cells. (C) GO analyses the biological processes and related diseases in which differential genes are enriched. (D) KEGG analyzed the related pathways of differential gene enrichment.

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