Modification of a natural diterpene and its antitumor mechanism: Promoting apoptosis, suppressing migration, and inhibiting angiogenesis

2.1 Synthesis of TPP derivative

2.2 Cell culture and related materials and reagents

A549, HepG2, and HeLa cells were cultured under the standard conditions required for their growth. Materials and reagents utilized in the biological experiments are described in the Supplementary data.

2.3 Cytotoxic activity assessment

The influence of the tested compounds on cancer cell survival was examined using the MTT assay (Zhang et al., 2021a).

2.4 Apoptosis analysis

The apoptosis of A549 cells affected by 1b was evaluated utilizing an Annexin V-FITC apoptosis kit, as reported previously (Zhang et al., 2022a). A549 cells were inoculated in 12-well plates (1 × 10⁵ cells per well) and cultured for 24 h. Subsequently, the A549 cells were treated with 1b (2, 4, and 8 μM) for 48 h. Then, the cells were gathered, washed with PBS twice, resuspended in Annexin V-FITC binding buffer (Beyotime, Shanghai, China), and stained with 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI). After 20 min of incubation at room temperature in the absence of light, the BD LSRFortessa flow cytometer (BD Biosciences) was employed to detect cell apoptosis. The percentages of apoptotic cells were obtained by analyzing the data via FlowJo software (FlowJo LLC, Ashland, OR, USA).

2.5 Mitochondrial membrane potential measurement

The impact of 1b on the mitochondrial membrane potential (Δψ_m) in A549 cells was assessed using the JC-1 assay, and the state of JC-1 (monomer or aggregates) was detected through flow cytometry (Zuo et al., 2022; Li et al., 2023). After inoculating A549 cells into 12-well plates (1 × 10⁵ cells per well) for 24 h incubation, different doses of 1b (3, 6, and 12 μM) were administered. After 48 h of culture, the cells were gathered, rinsed twice with chilled PBS, and then centrifuged (4 ℃, 2060 rpm, 5 min) to remove the PBS. Subsequently, the cell pellets were resuspended in 500 μL of JC-1 staining solution and incubated at 37 °C for 20 min in the dark. Then, the cells were subjected to centrifugation (4 °C, 2660 rpm, 5 min) to discard the supernatant, rinsed twice with PBS, and centrifuged again to eliminate the remaining PBS. Eventually, the cells were resuspended in 1 mL of pre-warmed PBS and promptly analyzed using a flow cytometer.

2.6 Determination of ROS production

The levels of ROS in A549 cells affected by 1b were measured using the DCFH-DA probe, which can be ingested by cells and undergo oxidation by ROS to produce DCF with fluorescence (Zhang et al., 2021b; Li et al., 2022). Briefly, A549 cells were seeded into 12-well plates (1 × 10⁵ cells/well) and then treated with 1b (2, 4, and 8 μM) for 48 h. Subsequently, the cells were gathered and co-incubated with the DCFH-DA (10 µM) probe for 20 min at 37 ℃ in the dark. After being rinsed three times with serum-free DMEM, the cells were suspended and examined utiulizing a flow cytometer (BD Biosciences).

2.7 Cell cycle analysis by flow cytometry

The cell cycle population affected by 1b was analyzed using flow cytometry (Zhang et al., 2022b; Li et al., 2021). In summary, A549 cells were seeded into 12-well plates (2 × 10⁵ cells per well) and cultured for 24 h. Following exposure to 1b (2, 4, and 8 μM) for 48 h, A549 cells were collected, rinsed twice with pre-cooled PBS, and then fixed in 70 % ethanol at 4 ℃ for 12–24 h. Subsequently, the harvested cells were rinsed with PBS twice and stained with PI staining buffer containing RNase (Beyotime, Shanghai) for 30 min in an incubator. Then, flow cytometry analysis was performed immediately, and the data were handled using ModFit LT software.

2.8 Wound-healing assay

The inhibition of 1b on the migration of A549 cells was examined utilizing a scratch test (Zhao et al., 2022; Lin et al., 2022). Briefly, A549 cells were seeded into a 6-well plate (5 × 10⁵ cells/well). After reaching 90 % fusion, the wound was scratched out with a sterile pipette tip. Then, different concentrations of 1b were added and treated for 48 h. The scratches at 0 h and 48 h were viewed under the microscope and photographed. The scratch area was determined by ImageJ software, and the cell mobility was calculated.

2.9 Western blotting analysis

Western blotting was executed as previously reported (Zhao et al., 2022; Li et al., 2023). The specifics are offered in the Supplementary data.

2.10 Zebrafish experiments

The protocols pertaining to zebrafish experiments received approval from the Animal Ethics Committee of Nankai University. The inhibition of 1b on angiogenesis was assessed utilizing transgenic zebrafish Tg(fli1:EGFP) as previously described (Bao et al., 2021; Li et al., 2021). In brief, transgenic zebrafish embryos were obtained by mating with Tg(fli1:EGFP) transgenic adult zebrafish and maintained for 6 h at 28.5 ℃. Subsequently, the embryos were randomly transferred into a 12-well plate with 15 embryos per well, exposed to 1b (1.25, 2.5, and 5 µM) and sunitinib malate (2 µM), and cultured for another 48 h at 28.5 ℃. Ultimately, 0.02 % tricaine was used to sedate the embryos, and Leica TCS SP8 confocal microscope (Wetzlar, Germany) was used to take pictures of the developing dorsal longitudinal anastomotic vessels (DLAVs) and intersegmental vessels (ISVs). The length of ISVs was quantified employing ImageJ software (NIH, Bethesda, Maryland, USA). For the in vivo anticancer assessment of 1b, zebrafish xenografts were utilized, and the experiments were carried out as reported previously (Zhou et al., 2021; Li et al., 2022). The details are appended in the Supplementary data.

2.11 Statistical analysis

Each experiment was performed at least three times. Data are shown as mean ± SD. Statistical evaluation was executed utilizing GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). The differences among groups were determined by one-way ANOVA multiple comparisons. A probability of *P < 0.05, ^**P < 0.01, and ^***P < 0.001 disclosed statistically significant values.

3.1 Molecule design and synthesis

As illustrated in Scheme 1, the hydroxy group in the structure of 3,4-seco-sonderianol was utilized to design and synthesize a TPP-linked 3,4-seco-sonderianol derivative (1b). The crucial intermediate (1a) was obtained by etherifying the C-12 hydroxy group in 3,4-seco-sonderianol with 1,5-dibromopentane. Finally, the target compound 1b was obtained by reacting it with triphenylphosphine and isolated with a yield of 14 %. According to the ¹H and ¹³C NMR spectra, as well as the HRESIMS data, the target molecule 1b was confirmed.

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Scheme 1

Synthesis of target derivative 1b. Reaction conditions: (a) 1,5-dibromopentane, acetone, K₂CO₃, 65 °C, reflux; (b) TPP, toluene, 80°C, reflux 12 h.

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3.2 1b suppressed the proliferation of cancer cells

Compound 1 was isolated and identified as a natural diterpene from the medicinal plant T. howii. In the following screening experiments, three carcinoma cell lines (A549, HepG2, and HeLa cells) were utilized, and IC₅₀ values were calculated to assess the effectiveness of compounds. Etoposide served as a positive control. The results are collated in Table 1. Compound 1 was found to have limited effects on the survival of tumor cells. In order to enhance the anticancer effects, compound 1 was modified to afford a new compound (1b), which was then subjected to MTT assays. The results in Table 1 demonstrated that the cytotoxicity of the modified molecule 1b was significantly improved by about 4–8 times compared to compound 1. Moreover, among the three human cancer cell lines, 1b had the greatest inhibition on the proliferation of A549 cells, surpassing the effectiveness of etoposide. Hence, based on the sensitivity of the compound to these cancerous cells, A549 cells were utilized to investigate the mechanism of action of 1b in the following experiments.

3.3 1b triggered programmed cell death in A549 cells

Multiple investigations have revealed a link between apoptosis and the onset and progression of tumors (Wong, 2011). To investigate the potential relationship between the inhibition of compound 1b on A549 cell growth and apoptosis, the proportion of cells undergoing apoptosis after treatment with 1b was assessed by flow cytometry (Zuo et al., 2022). As depicted in Fig. 1, the proportion of apoptotic cells rose as the concentration of 1b increased. Specifically, the percentage increased from 7.33 % in the control group to 18.50 % at a dose of 2 μM, 24.80 % at 4 μM, and 29.07 % at 8 μM. The findings demonstrated that 1b caused the programmed cell death in A549 cells in a way that depended on the concentration.

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Fig. 1

Apoptosis triggered by 1b in A549 cells. A549 cells were exposed to 1b for 48 h. DMSO served as a negative control. Apoptotic levels were evaluated using Annexin V FITC/PI double labeling and flow cytometry. (A) Flow cytometry analysis after treatment with 1b. (B) Histogram of the apoptosis cells after treatment with 1b. All values are expressed as the mean ± SD. ^**P < 0.01 and ^***P < 0.001 vs. the control group.

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3.4 1b impacted the cell cycle

A continuous cellular cycle is a necessary condition for ensuring the completion of cell proliferation. The impact of 1b on cell cycle arrest was examined to further clarify the mechanism of apoptosis induced by 1b (Qiu et al., 2022). As depicted in Fig. 5, the percentage of A549 cells in the G0/G1 and S stages decreased gradually, while the proportion in the G2/M stage increased after exposure to 1b (2, 4, and 8 μM) for 48 h, in comparison to the control group. Additionally, the proportion in the G2/M stage rose from 1.41 % (control group) to 3.52 % (2 μM), 7.07 % (4 μM), and 10.98 % (8 μM), indicating that 1b had the capacity to arrest the A549 cell cycle in the G2/M stage.

3.5 1b stimulated the generation of ROS in A549 cells

ROS significantly impact several metabolic processes and physiological responses, e.g., cell growth, differentiation, mobility, and programmed cell death. Apoptosis is usually triggered by a substantial elevation of ROS in mitochondria, and this aberrant increase in ROS frequently causes cell death (Zhang et al., 2019). To detect ROS levels, the DCFH-DA fluorescent probe was used, and the alteration in fluorescence intensity was quantified using flow cytometry to assess the impact of 1b on the amount of ROS in A549 cells. As displayed in Fig. 4, the administration of 1b resulted in elevated levels of ROS in A549 cells. Specifically, the ROS levels were 1.64 times higher at a concentration of 2 μM, 2.76 times higher at 4 μM, and 6.99 times higher at 8 μM, in comparison to the control group. Thus, it can be inferred that 1b induced apoptosis by increasing the amounts of intracellular ROS, thereby demonstrating its anticancer effects.

3.6 1b disrupted mitochondrial membrane potential

The drop in mitochondrial membrane potential (Δψ_m) is a noteworthy occurrence during the initial phases of apoptosis (Shi et al., 2021). The TPP molecule is a primary structural unit that specifically targets mitochondria (Battogtokh et al., 2018; Kafkova et al., 2023). To examine the capacity of the TPP-modified substance (1b) to reduce Δψ_m and trigger programmed cell death, the fluorescent probe JC-1 was employed for staining, and the alteration in Δψ_m was then measured by flow cytometry. When the mitochondrial membrane potential (Δψ_m) is elevated, JC-1 gathers in the inner compartment of mitochondria to form clusters, resulting in the emission of red fluorescence. At a low mitochondrial membrane potential (Δψ_m), JC-1 is present in the cytoplasm as a single unit and may emit green fluorescence (Zuo et al., 2022). The results in Fig. 2A-B demonstrated that the most noticeable drop in Δψ_m occurred when the concentration of compound 1b was 12 μM. The JC-1 monomer percentage rose to 20.27 %, while the proportion of JC-1 aggregates declined to 78.03 %. Furthermore, the ratio of JC-1 aggregates to monomers in Fig. 2C reduced from 15.70 % (control group) to 3.85 % (12 μM). The measurement of Δψ_m revealed that 1b can reduce Δψ_m and trigger apoptosis in A549 cells.

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Fig. 2

Effects of 1b on mitochondrial membrane potential (Δψ_m). (A) Cells were exposed to 1b for 48 h and then stained with JC-1 dye. ∆ψ_m was detected by flow cytometry. (B) The percentage of cells with red aggregates and cells with green monomers. (C) The ratio of JC-1 aggregates/monomers. All values are expressed as the mean ± SD. ^***P < 0.001 vs. the control group.

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3.7 1b influenced the levels of proteins associated with apoptosis

The loss of Δψ_m indicates substantial mitochondrial damage, which can trigger the intrinsic mitochondrial apoptotic pathway (Zhang et al., 2019). Bax proteins reassemble on the surface of the mitochondria to form membrane channels, which increases the permeability of the mitochondrial membrane and facilitates the diffusion of the pro-apoptotic factor, cytochrome c, into the cytoplasm. Cytochrome c binds to apoptotic protease-activating factor 1 (Apaf-1) to form a complex and then stimulates caspase, initiating the caspase cascade that ultimately leads to apoptosis (Shi et al., 2021; Wang et al., 2023). To further address the molecular mechanism through which 1b prompted apoptosis in A549 cells, the levels of Bax and the activation of caspases were examined by Western blotting analysis. The data revealed that the administration of 1b led to a dose-dependent increase in the levels of Bax and cleaved caspase-3, while the amounts of caspase-3 and caspase-9 decreased in a dose-dependent pattern (Fig. 3). Collectively, the findings unequivocally demonstrated that 1b triggered apoptosis of A549 cells via the mitochondrial route.

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Fig. 3

Effects of 1b on the levels of apoptosis-related proteins. Cells were exposed to 1b for 48 h. (A) Western blotting analysis of caspase-9, caspase-3, Bax, and cleaved caspase-3. (B-E) Quantitative analysis of the Western blotting (n = 3). *P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. the control group.

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Fig. 4

Effects of 1b on ROS generation. A549 cells were exposed to 1b for 48 h. (A) ROS accumulation in A549 cells determined by flow cytometry using DCFH-DA staining. (B) Relative ROS levels in A549 cells. All values are expressed as the mean ± SD. ^***P < 0.001 vs. the control group.

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Fig. 5

Effects of 1b on cell cycle distribution. A549 cells were treated with 1b for 48 h. DMSO served as a negative control. (A) Cell cycle distribution analyzed using flow cytometry. (B) Data processing for cell cycle distribution. All values are expressed as the mean ± SD. *P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. the control group.

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3.8 1b regulated STAT3 signaling pathway

STAT3 serves as a convergence point for many signal transduction pathways in malignancies, playing a vital role in the onset and advancement of cancerous tumors (Johnston and Grandis, 2011). Overactivation of STAT3 may cause an aberrant increase in the levels of genes that regulate the cell cycle, leading to cell proliferation. This process usually initiates tumor proliferation and promotes tumor progression (Fathi et al., 2018; Thilakasiri et al., 2021). To assess the suppressive impact of 1b on the STAT3 signaling pathway, A549 cells were incubated with 1b, and the cellular extracts were then examined by Western blotting. The treatment with 1b led to a reduction in the phosphorylation of STAT3 at tyrosine 705, as depicted in Fig. 6A. Specifically, when the concentrations of 1b reached 4 and 8 μM, the expression of p-STAT3 (Tyr705) dropped dramatically, almost completely disappearing. Simultaneously, the levels of STAT3 remained consistent despite variations in the concentration of 1b, as depicted in Fig. 6C-D. Furthermore, as the dose of 1b rose, there was a significant reduction in the level of cyclin D1 downstream of the STAT3 signaling pathway (Fig. 6E). These results demonstrated that 1b selectively inhibited the STAT3 signaling pathway.

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Fig. 6

Regulation of the STAT3 and FAK signaling pathways by 1b. (A-B) Western blotting analysis of the levels of STAT3, p-STAT3 (Tyr705), cyclin D1, FAK, p-FAK (Tyr397), and MMP-2. (C-H) Quantitative analysis of the relative protein expression levels of multiple proteins. The results are expressed as the mean ± SD. ^**P < 0.01, ^***P < 0.001 vs. the control group.

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3.9 1b suppressed cancerous cell migration and influenced the FAK signaling pathway

Apart from uncontrolled growth, migration is a crucial feature of tumors. To investigate the impact of 1b on the migration of cancerous cells, a wound-healing assay was undertaken (Zhai et al., 2023; Zuo et al., 2022). Following exposure to 1b, the migration of A549 cells was substantially impeded as shown in Fig. 7. The mobility of A549 cells dropped from 76.00 % (control group) to 54.91 % (2 μM), 29.34 % (4 μM), and 14.00 % (8 μM) as the concentration of 1b rose. The scratch test confirmed that compound 1b had the capacity to inhibit the migration of cancer cells.

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Fig. 7

Influence of 1b on the migration of A549 cells.  (A) Images of wound healing photographed at 0 h and 48 h (scale bar: 100 μm). (B) Statistical analysis of the cell migration rate. DMSO served as a negative control. The results are presented as the mean ± SD. ^***P < 0.001 vs. the control group.

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Numerous investigations have revealed that the FAK signaling pathway is intimately linked to cancer cell migration. FAK is a crucial non-receptor intracellular tyrosine kinase, which can regulate many life processes such as cell cycle, cell invasion, and metastasis (Qiu et al., 2022). Moreover, FAK can regulate the expression of matrix metalloproteinases (MMPs) through integrin-mediated signaling pathways (Qiao et al., 2022), thereby controlling the invasive and metastatic behavior of cancerous cells (Määttä et al., 2004). Through Western blotting experiments, the impact of 1b on the levels of FAK and its associated proteins was assessed. As depicted in Fig. 6B and F-H, the presence of 1b did not significantly alter the cellular FAK expression, but resulted in a dose-dependent decrease in the expressions of p-FAK (Tyr397) and MMP-2. This suggested that 1b may impede A549 cell migration through suppressing the FAK route.

3.10 1b displayed anti-angiogenetic properties

Neovascularization is essential for the formation, growth, and spread of tumors by supplying nutrients to tumor cells. Therefore, blocking angiogenesis is a critical aspect of preventing tumor development (Viallard and Larrivée, 2017). The transgenic zebrafish Tg (fli1:EGFP) model enables the precise observation of blood vessel formation in vivo owing to the particular expression of green fluorescent protein (EGFP) (Delov et al., 2014; Gawrońska-Grzywacz et al., 2022). Therefore, the influence of 1b on angiogenesis was assessed using transgenic zebrafish. From Fig. 8, the embryos in the control group exhibited normal development with fully developed blood vessels. However, when exposed to varying concentrations of 1b, the growth of ISVs and DLAVs was significantly impaired. The control group had an average length of ISVs of 3541.44 μm. In each group treated with 1b, the average length of ISVs reduced in a dosage-dependent way (2890.83 ± 86.13 μm at 1.25 μM, 2559.87 ± 70.12 μm at 2.5 μM, 1735.49 ± 167.66 μm at 5 μM). At a dosage of 5 μM, the anti-angiogenetic effects of 1b were comparable to those of sunitinib malate, and there was no statistically noteworthy disparity in the average ISV duration between the two groups.

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Fig. 8

Anti-angiogenetic activity of 1b in a transgenic zebrafish model. The embryos were treated with 1b and sunitinib malate for 48 h. The development of ISVs and DLAVs was observed, and the length of ISV vessels was quantified using the ImageJ program. (A) Representative images of zebrafish embryos treated with vehicle, 1b, and sunitinib malate. The absence and breakage of ISVs are indicated by red arrows. (B) The average total length of ISVs of zebrafish after treatment with various concentrations of 1b. The results are expressed as the mean ± SD. *P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. the control group.

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3.11 In vivo anticancer efficacy of 1b

Compound 1b demonstrated excellent in vitro activity, as shown by the cell-level activity test and mechanistic examinations. To explore its potential application as an anticancer agent, the in vivo anticancer efficacy of 1b was further examined (Zhou et al., 2021). CM-DiI-labeled A549 cells were microinjected into the yolk sacs of 48 hpf embryos. The transplanted tumor cells displayed red fluorescence and were observable under confocal microscopy. As exhibited in Fig. 9A, strong fluorescence was observed in the control group. In addition to the fluorescence at the primary focal point (yolk sac), numerous metastatic fluorescent focal points were also observed. After administering 1b, the fluorescence intensity decreased gradually with increasing concentration, and the number of transferred fluorescence foci also decreased. After quantitative treatment by ImageJ software, it was determined that the inhibition rates of 1b on tumor cell proliferation were 22.50 % (1.25 μM), 40.56 % (2.5 μM), and 68.64 % (5 μM), respectively (Fig. 9B). Furthermore, the migration rate of tumor cells decreased in a dose-responsive way, from 72.00 % (1.25 μM), 50.67 % (2.5 μM), to 30.67 % (5 μM). When the concentration reached 5 μM, the inhibition was superior to that of the positive control, etoposide. The findings revealed that 1b can impede the proliferation and spread of A549 cells in zebrafish.

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Fig. 9

In vivo anticancer efficacy of 1b in zebrafish xenografts. CM-DiI-stained A549 cells were transplanted into 2 dpf zebrafish embryos via microinjection. 4 h later, tumor-bearing embryos were exposed to 1b and etoposide for 48 h. (A) Representative images of the relative intensity and distribution of the red fluorescence. (B) The fluorescence intensity of the tumor xenografts, representing the number of A549 cells. (C) The fluorescent area of the tumor xenografts, representing A549 cell metastasis. The results are expressed as the mean ± SD. *P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. the control group.

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