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Baculovirus-mediated endostatin and angiostatin activation of autophagy through the AMPK/AKT/mTOR pathway inhibits angiogenesis in hepatocellular carcinoma

  • Tingting Wei , Jiajie Cheng , Yonggan Ji , Xue Cao , Shuqin Ding , Quanxia Liu and Zhisheng Wang EMAIL logo
Published/Copyright: July 29, 2024
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Open Life Sciences
From the journal Open Life Sciences Volume 19 Issue 1

Abstract

Hepatocellular carcinoma (HCC) is a highly vascularized carcinoma, and targeting its neovascularization represents an effective therapeutic approach. Our previous study demonstrated that the baculovirus-mediated endostatin and angiostatin fusion protein (BDS-hEA) effectively inhibits the angiogenesis of vascular endothelial cells and the growth of HCC tumors. However, the mechanism underlying its anti-angiogenic effect remains unclear. Increasing evidence suggests that autophagy has a significant impact on the function of vascular endothelial cells and response to cancer therapy. Hence, the objective of this research was to investigate the correlation between BDS-hEA-induced angiogenesis inhibition and autophagy, along with potential regulatory mechanisms. Our results demonstrated that BDS-hEA induced autophagy in EA.hy926 cells, as evidenced by the increasing number of autophagosomes and reactive oxygen species, accompanied by an upregulation of Beclin-1, LC3-II/LC3-I, and p62 protein expression. Suppression of autophagy using 3-methyladenine attenuated the functions of BDS-hEA-induced EA.hy926 cells, including the viability, proliferation, invasion, migration, and angiogenesis. Moreover, BDS-hEA induced autophagy by downregulating the expression of CD31, VEGF, and VEGFR2, as well as phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR), while concurrently upregulating phosphorylated AMP-activated protein kinase (p-AMPK). The in vivo results further indicated that inhibition of autophagy by chloroquine significantly impeded the ability of BDS-hEA to suppress HCC tumor growth in mice. Mechanistically, BDS-hEA prominently facilitated autophagic apoptosis in tumor tissues and decreased the levels of ki67, CD31, VEGF, MMP-9, p-AKT, and p-mTOR while simultaneously enhancing the p-AMPK expression. In conclusion, our findings suggest that BDS-hEA induces autophagy as a cytotoxic response by modulating the AMPK/AKT/mTOR signaling pathway, thereby exerting anti-angiogenic effects against HCC.

Keywords: hepatocellular carcinoma; baculovirus; endostatin-angiostatin; anti-angiogenesis; autophagy

1 Introduction

Hepatocellular carcinoma (HCC) is a prevalent human malignant tumor, constituting about 80% of all cases of primary liver cancer [1]. The current therapeutic approaches continue to encounter challenges attributed to the highly aggressive metastatic propensity of HCC [1]. The growth and metastasis of HCC are primarily driven by angiogenesis, resulting in a high incidence of hematogenous metastases and exceptionally elevated mortality [2]. Consequently, inhibition of neovascularization has become one of the effective strategies to impede tumor progression and dissemination.

Currently, numerous endogenous anti-angiogenic agents, such as endostatin, tumstatin, and angiostatin, have been extensively investigated and their anti-tumor angiogenesis effects were confirmed [3]. Nevertheless, the therapeutic efficiency of utilizing these protein-based inhibitors remains limited due to their short half-life and the potential for development of drug resistance with long-term administration [4]. In recent years, the sustained expression of foreign genes through gene therapy appears to be a promising approach for overcoming these limitations. Previously, we developed a bivalent baculovirus vector that not only resists complement system inactivation but also continuously expresses foreign genes. Expression of the endostatin (collagen α1-chain XVIII C-terminal hydrolytic fragment) and angiostatin (amino-terminal proteolytic fragment of plasmin) fusion protein using this vector (BacSC-DAF-SB-T2ChEA, abbreviated as BDS-hEA) exhibited enhanced and sustained inhibition of vascular endothelial cell angiogenesis, proliferation, and migration, along with significant suppression of HCC tumor growth in mice [5]. Moreover, when combined with gemcitabine treatment, the synergistic therapeutic effect was more pronounced than a single application [6]. Nevertheless, the precise mechanism underlying the anti-tumor angiogenic effect of BDS-hEA remains unclear.

Autophagy, a self-protective mechanism developed by cells during long-term evolution, is a process in which damaged substances undergo self-degradation through lysosomes in response to external interference or disruption of cellular homeostasis [7]. The process of autophagy is an intricate biological phenomenon, involving multiple signaling pathways in its regulation. For example, the mammalian target of rapamycin (mTOR), a cellular nutrient and energy level sensor, negatively regulates autophagy, while protein kinase B (AKT) and AMP-activated protein kinase (AMPK), acting as upstream regulators, positively and negatively regulate mTOR levels, respectively [8]. Emerging findings indicate a potential association between autophagy and the angiogenic activity observed in different types of cancers [9]. However, there appears to be contradictory evidence regarding the involvement of autophagy in angiogenesis. For instance, Sun et al. confirmed that arsenic trioxide effectively inhibited angiogenesis and induced autophagy in human umbilical vein endothelial cells (HUVECs), and inhibition of autophagy significantly reversed their anti-angiogenic effect [10]. Interestingly, Xue et al. reported that lenvatinib could induce autophagy in thyroid cancer cells through the PI3K/AKT/mTOR pathway, and autophagy suppression enhanced the cytotoxicity and anti-angiogenic capacity of lenvatinib [11]. Nevertheless, the precise role and regulatory mechanism of autophagy underlying BDS-hEA-induced anti-angiogenesis have not been completely clarified.

In this research, we explored the relationship between BDS-hEA-induced inhibition of angiogenesis and autophagy, elucidating their underlying molecular mechanisms. Our findings demonstrate that BDS-hEA induces autophagy as a cytotoxic response by modulating the AMPK/AKT/mTOR pathway. These findings highlight the potential of targeting autophagic response to enhance the therapeutic efficacy of angiogenesis inhibitors.

2 Materials and methods

2.1 Reagents

Antibodies against VEGFR2 and VEGF were obtained from Santa Cruz (California, USA); antibody against LC3B was purchased from Abmart (Shanghai, China); antibodies against Beclin-1, p62, CD31, Ki67, AKT, p-AKT, mTOR, p-mTOR, AMPK, p-AMPK, and HRP-labeled rabbit/mouse IgG were procured from Abcam (Cambridge, USA); antibody against MMP-9 was obtained from Proteintech (Wuhan, China); and antibodies against β-actin and FITC-labeled rabbit/mouse IgG were procured from ZSGB-BIO (Beijing, China). TUNEL red fluorescence in situ apoptosis detection kit was purchased from Vazyme (Nanjing, China). Reactive oxygen species (ROS) detection kit was obtained from Solarbio (Beijing, China). 3-Methyladenine (3-MA) was obtained from APExBIO (Houston, USA). Chloroquine (CQ) was purchased from MCE (New Jersey, USA).

2.2 Viruses and cells

The construction and transduction methods of recombinant baculovirus BDS-hEA have been described previously [5]. EA.hy926 cells (human umbilical vein endothelial fusion cell line) and HepG2 cells (human hepatocellular carcinoma cell line) were grown in DMEM (Sigma, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin solution (Gibco, USA) in a 5% CO2 incubator with a temperature of 37°C.

2.3 Monodansylcadaverine (MDC) and mGFP-LC3-adenovirus staining

EA.hy926 cells with or without 3-MA (4 mM) in the medium were incubated in 12-well plates (7 × 104 cells/well) overnight. Subsequently, the cells were transduced with BDS-hEA for 48 h at different multiplicity of infection values (MOIs) and then stained with 200 μL of MDC solution (Solarbio, Beijing, China) in dark at room temperature for 30 min. Alternatively, the EA.hy926 cells were exposed to mGFP-LC3 adenovirus (HANBIO, Shanghai, China) at a concentration of 10 MOI for 24 h prior to transduction with BDS-hEA for another 48 h. Finally, the formation and visualization of autophagosomes were observed under an inverted fluorescence microscope.

2.4 ROS detection

EA.hy926 cells were grown in 12-well plates (7 × 104 cells/well) overnight and then tansduced with BDS-hEA at MOI values of 100 and 400 for 48 h. Subsequently, each well was treated with 200 μL of diluted DCFH-DA fluorescent probe (10 μmol/L), followed by incubation at 37°C for 20 min. The cells were then observed and documented under an inverted fluorescence microscope.

2.5 Cell viability assay

EA.hy926 cells with or without 3-MA (4 mM) in the medium were incubated in 96-well plates (7 × 103 cells/well) overnight, followed by transduction with BDS-hEA (MOI 400) for 48 h. Afterward, fresh DMEM supplemented with 10% CCK-8 solution (Beyotime, Shanghai, China) was introduced into each well and incubated for 30 min at 37°C. The microplate reader (Multiskan GO, Thermo, USA) was then utilized to measure the optical density (OD) value at a wavelength of 450 nm for every group that had five technical replicates. Cell viability (%) was calculated as follows: (ODtreatment − ODblank)/(ODcontrol − ODblank) × 100%.

2.6 Colony formation assay

EA.hy926 cells with or without 3-MA (4 mM) were transduced with BDS-hEA (MOI: 400) for 48 h, and then the cells in each group were trypsinized and inoculated into 6-well plates (700 cells/well). The cells were cultured for approximately 2 weeks until visible colonies became apparent. Following that, the colonies were fixed using 4% paraformaldehyde, dyed with 0.1% crystal violet, and photographed by an inverted microscope.

2.7 Cell wound healing assay

EA.hy926 cells with or without 3-MA (4 mM) were grown in six-well plates (7 × 105 cells/well) overnight. Subsequently, the cells were transduced with BDS-hEA (MOI: 400) for 48 h. When the cells had completely covered the bottom surface of each well, a gentle scraping was performed utilizing a 200 μL pipette tip. Following that, the medium was changed to DMEM supplemented with 1% FBS. Immediate and subsequent photographs were captured at a time interval of 24 h post-incubation. The migration ratio was then calculated by comparing the average migrated area with the initial scratched area, utilizing ImageJ software (NIH, USA).

2.8 Cell invasion assay

EA.hy926 cells with or without 3-MA were transduced with BDS-hEA (MOI 400) for 48 h, and then the cells were resuspended (2 × 104 cells/well) in FBS-free DMEM and seeded in the upper chamber (precoated with a matrix solution containing 0.5 mg/mL from Corning, NY, USA) of a 24-well transwell plate with a pore diameter of 8 μM. The lower chamber contained DMEM enriched with 10% FBS. After 24 h, the upper chamber non-invasive cells were scraped off with a cotton swab, while the invasive cells situated beneath the membrane were immobilized using a 4% paraformaldehyde and dyed utilizing a 0.1% crystal violet solution. Finally, images were taken using an inverted microscope.

2.9 Tubule formation assay

EA.hy926 cells with or without 3-MA were transduced with BDS-hEA (MOI 400) for 48 h, and then the cells were resuspended and seeded in precoated matrigel (50 μL/well) in 96-well plates (3 × 104 cells/well) for 4 h at 37°C. After that, the formation of tubule networks was observed by an inverted fluorescence microscope (100×). Quantification was conducted by utilizing ImageJ software to calculate the cumulative length of master segments in five fields that were randomly chosen.

2.10 Western blot assay

EA.hy926 cells with or without 3-MA were cultured in a 10 cm cell culture dish and subsequently transduced with BDS-hEA at different MOI values for 48 h. Afterward, the cells were harvested, lysed, and the protein concentration was determined using the BCA kit (KeyGen, Nanjing, China). Equivalent quantities of protein were separated by 10–12% SDS-PAGE gels and then moved onto a PVDF membrane (Millipore, MA, USA). Subsequently, the bands were blocked using skim milk for a duration of 1 h and incubated overnight at 4°C with primary antibodies. Following that, the bands were exposed to secondary antibodies for 1 h at room temperature. Next, the bands were exposed to an enhanced chemiluminescence (ECL) solution (Thermo, USA) for 1 min in darkness before being subjected to analysis and quantification utilizing the gel imaging system (Bio-Rad, USA) and ImageJ software (NIH, USA), respectively.

2.11 HCC xenograft model in mice

Twenty male BALB/c nude mice (4–6 weeks, Weitong Lihua, Beijing, China) were housed in a standardized SPF-grade barrier facility. The animal experiment adhered to the ethical guidelines that were approved by the Ethics and Welfare Committee of Ningxia Medical University (IACUC-NYLAC-2021-135). HepG2-EGFP and luciferase cells (EGFP = enhanced green fluorescent protein), which stably expressed EGFP and luciferase through lenti-CMV-EGFP and luciferase infection, were introduced into each mouse’s right dorsal region via subcutaneous injection using 100 μL of PBS containing 2 × 106 cells. After the tumor volume grew to around 100 mm3, a random allocation was made to divide the mice into four groups with five animals per group for subsequent injections as follows: control group (intratumoral injection of an equal volume of PBS in a 7-day interval four times); BDS-hEA group (intratumoral injection of 1 × 108 pfu/mouse in a 7-day interval four times); CQ group (intraperitoneal injection of 50 mg/kg/mouse every 2 days); and BDS-hEA + CQ group (identical dose and frequency as previously). The measurement of tumor volume was conducted at 3-day intervals using a dial caliper, and the tumor growth status was real-time-monitored every 7 days using a small animal live imaging system (IVIS LUMINA III, USA). On day 28 post-administration, all mice were euthanized using a CO2 inhalation euthanasia device (SMQ-II-Q, Shanghai, China), and tumor tissues were stripped for the following tests. (Only the doser has access to information about animal grouping throughout the entire process.)

  1. Ethical approval: The research related to animal use complied with all the relevant national regulations and institutional policies for the care and use of animals.

2.12 Immunohistochemistry staining

Tumor tissues were treated with 4% paraformaldehyde, followed by embedding in paraffin. Subsequently, the paraffin sections were processed for dewaxing, hydration, antigen retrieval, and sealing according to the immunohistochemistry kit (ZSGB-BIO, Beijing, China). The tissue sections were then subjected to overnight incubation at 4°C with each of the primary antibodies Beclin-1, LC3B, p62, p-AKT, p-mTOR, and p-AMPK. Afterward, the sections were exposed to secondary antibodies conjugated with HRP for 1 h at ambient temperature. Diaminobenzidine (DAB) was utilized as a chromogen to visualize the antigen–antibody complexes, and hematoxylin was employed as a counterstain. The photographs were captured using a fluorescence microscope (Ni-U, Nikon, Japan), then five photos were randomly selected, and the integrated optical density (IOD)/area was measured utilizing Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA) to evaluate the positive expression rate of each section.

2.13 Immunofluorescence and TUNEL staining

The fresh tumor specimens were embedded in OCT solution and subsequently prepared as frozen sections. Antigen retrieval and sealing procedures were then conducted according to the instructions provided in the immunofluorescence kit (ZSGB-BIO, Beijing, China). The sections were subsequently subjected to overnight incubation at 4°C with each of the primary antibodies CD31, VEGF, Ki67, and MMP-9. Following that, FITC-conjugated corresponding species secondary antibodies were applied to incubate at room temperature for 1 h, followed by counterstaining with DAPI in darkness for 10 min. The TUNEL assay was conducted following the standard protocol provided by Vazyme (Nanjing, China) to detect tissue apoptosis. The images were photographed and analyzed as described above.

2.14 Statistical analysis

The data obtained from this study were subject to one-way analysis of variance using SPSS 26.0 software (IBM, NY, USA). The results were based on a minimum of three independent experiments. All the experiments were repeated at least three times. Statistical significance was determined at p < 0.05, p < 0.01, and p < 0.001.

3 Results

3.1 BDS-hEA induces autophagy in vascular endothelial cells

Previous research has demonstrated the significant inhibitory effects of BDS-hEA on the viability and tubule formation of vascular endothelial cells [5]. However, the underlying mechanism behind this phenomenon remains unclear. Extensive research works have suggested a strong correlation between tumor angiogenesis and autophagy [9]. To investigate whether BDS-hEA can induce autophagy in vascular endothelial cells, we examined the changes in autophagic morphology by tracking the number of autophagosomes through staining with EGFP-LC3-adenovirus and MDC. Figure 1a demonstrates a dose-dependent rise in the number of EGFP-LC3-adenovirus-labeled green spots and MDC-labeled blue cells within the BDS-hEA group, as compared to the control group. After pretreatment with 3-MA to inhibit autophagosome formation, followed by transduction with BDS-hEA (MOI 400), a noticeable decrease in the quantity of fluorescent cells was found after combined treatment compared to BDS-hEA alone in EA.hy926 cells (Figure 1b). Studies have indicated that the initiation of autophagy potentially lead to the buildup of intracellular ROS [12]. Hence, an investigation was further conducted to assess the intracellular levels of ROS using the fluorescent probe 2ʹ7ʹ-dichlorodihydrofluorescein diacetate (DCFH-DA). The finding revealed a significant dose-dependent increase in ROS levels within the BDS-hEA group in comparison with the control group (Figure 1c). Additionally, the Beclin-1, p62, and LC3-II/LC3-I proteins related to autophagy were further assessed through western blot. The results depicted in Figure 1d indicated that there was a gradual increase in the expression of aforementioned proteins in the BDS-hEA group with increasing doses. However, when combined with 3-MA, a significant trend of protein reversal was observed in comparison to the BDS-hEA group (Figure 1e). These findings collectively confirm that BDS-hEA has the capability to trigger autophagy in EA.hy926 cells.

Figure 1 BDS-hEA induces autophagy in vascular endothelial cells. The EA.hy926 cells were transduced with BDS-hEA for 48 h with or without 3-MA. EGFP-LC3-adenovirus and MDC staining were used to detect the morphology of autophagy without (a) or with 3-MA (b). (c) ROS was detected by the DCFH-DA fluorescent probe. The Beclin-1, p62, LC3-I, and LC3-II proteins were analyzed by western blot without (d) or with 3-MA (e), with β-actin as a loading control. All the experiments were repeated at least three times. **p < 0.01 and ***p < 0.001 vs control group. ## p < 0.01 and ### p < 0.001 vs BDS-hEA group.
Figure 1

BDS-hEA induces autophagy in vascular endothelial cells. The EA.hy926 cells were transduced with BDS-hEA for 48 h with or without 3-MA. EGFP-LC3-adenovirus and MDC staining were used to detect the morphology of autophagy without (a) or with 3-MA (b). (c) ROS was detected by the DCFH-DA fluorescent probe. The Beclin-1, p62, LC3-I, and LC3-II proteins were analyzed by western blot without (d) or with 3-MA (e), with β-actin as a loading control. All the experiments were repeated at least three times. **p < 0.01 and ***p < 0.001 vs control group. ## p < 0.01 and ### p < 0.001 vs BDS-hEA group.

3.2 Blocking autophagy impairs the functionality of vascular endothelial cells induced by BDS-hEA

To further investigate the effect of BDS-hEA-induced autophagy on the function of vascular endothelial cells, we assessed the impact of 3-MA-mediated autophagy inhibition on BDS-hEA-induced EA.hy926 cell proliferation, invasion, and migration, as well as tubule formation and angiogenesis-related protein expression alterations. The results from CCK-8 and clone formation assays showed that there is a significant enhancement in the cell viability and proliferation ability within the BDS-hEA + 3-MA-treated group compared to the BDS-hEA group (Figure 2a and b). Furthermore, BDS-hEA + 3-MA significantly attenuated the migration, invasion, and angiogenic capacity of EA.hy926 cells treated with BDS-hEA, as demonstrated by the cell scratch, invasion, and tubule formation assays (Figure 2c and d). Additionally, we investigated the alterations in proteins associated with angiogenesis and found that BDS-hEA significantly suppressed the expression of CD31, VEGF, and VEGFR2 proteins. When combined with 3-MA, there was a significant reversal trend in comparison with the BDS-hEA group (Figure 2e). In combination, these results suggest that the inhibition of autophagy hinders the growth, invasion, migration, and angiogenesis in BDS-hEA-induced vascular endothelial cells.

Figure 2 Blocking autophagy impairs the functionality of vascular endothelial cells induced by BDS-hEA. The EA.hy926 cells were transduced with BDS-hEA (MOI, 400) for 48 h with or without 3-MA. (a) Cell viability was evaluated by the CCK-8 assay. (b) Cell proliferation ability was assessed by the colony formation assay. (c) Cell migratory and invasive capacities were evaluated by scratch and transwell assays. (d) Vascularization ability was measured by the tubule formation assay. (e) Detection of angiogenesis-related proteins after 3-MA intervention. The data from three repeated experiments were used for the statistical analysis. ***p < 0.001 vs control group. # p < 0.05, ## p < 0.01, and ### p < 0.001 vs BDS-hEA group.
Figure 2

Blocking autophagy impairs the functionality of vascular endothelial cells induced by BDS-hEA. The EA.hy926 cells were transduced with BDS-hEA (MOI, 400) for 48 h with or without 3-MA. (a) Cell viability was evaluated by the CCK-8 assay. (b) Cell proliferation ability was assessed by the colony formation assay. (c) Cell migratory and invasive capacities were evaluated by scratch and transwell assays. (d) Vascularization ability was measured by the tubule formation assay. (e) Detection of angiogenesis-related proteins after 3-MA intervention. The data from three repeated experiments were used for the statistical analysis. ***p < 0.001 vs control group. # p < 0.05, ## p < 0.01, and ### p < 0.001 vs BDS-hEA group.

3.3 BDS-hEA induces autophagy in vascular endothelial cells through modulation of the AMPK/AKT/mTOR signaling pathway

Numerous studies have confirmed that AMPK and AKT/mTOR pathways are implicated in the regulation of autophagy [13]. Therefore, we performed the western blot assay to investigate the potential involvement of this cascade in BDS-hEA-treated EA.hy926 cells. As depicted in Figure 3a, with the escalation of BDS-hEA dosage, an evident rise in the expression level of p-AMPK/AMPK was observed, whereas there was a notable decrease in p-AKT/AKT and p-mTOR/mTOR levels when compared to those in the control group. When 3-MA was added, the aforementioned protein levels were reversed in comparison to those observed in the BDS-hEA group alone (Figure 3b). Overall, BDS-hEA induces the autophagy of EA.hy926 cells by activating AMPK and suppressing AKT/mTOR pathways.

Figure 3 BDS-hEA induces autophagy in vascular endothelial cells through the modulation of the AMPK/AKT/mTOR signaling pathway. The pathway-related proteins were detected by western blot in EA.hy926 cells treated with BDS-hEA (MOI 100 or 400) for 48 h without (a) or with 3-MA (b), with β-actin as a loading control. Three repeated experiments were used for analysis. ***p < 0.001 vs control group; ### p < 0.001 vs BDS-hEA group.
Figure 3

BDS-hEA induces autophagy in vascular endothelial cells through the modulation of the AMPK/AKT/mTOR signaling pathway. The pathway-related proteins were detected by western blot in EA.hy926 cells treated with BDS-hEA (MOI 100 or 400) for 48 h without (a) or with 3-MA (b), with β-actin as a loading control. Three repeated experiments were used for analysis. ***p < 0.001 vs control group; ### p < 0.001 vs BDS-hEA group.

3.4 BDS-hEA inhibits the growth of HCC by inducing autophagy in tumor tissues

Our previous study has demonstrated that BDS-hEA can effectively inhibit the growth of HCC xenograft tumors in mice [5], but the relationship between its inhibitory effect and autophagy remains unclear. Therefore, we evaluated the inhibitory effect of BDS-hEA on HCC by creating a xenograft model of nude mice, before and after intervention with the autophagy inhibitor CQ. The results revealed a gradual enhancement in the fluorescence intensity of control tumors over time, whereas the BDS-hEA group exhibited significantly diminished fluorescence compared with the control group. Conversely, the BDS-hEA + CQ group showed an augmented fluorescence intensity compared to the BDS-hEA group, with a more pronounced reversal trend, particularly at 28 days post-administration (Figure 4a). Similar findings were also confirmed that the BDS-hEA group exhibited significant reductions in the tumor size, volume, and weight when compared remaining three groups. Notably, the BDS-hEA + CQ group showed a significant counteraction of these parameters in comparison to the BDS-hEA alone group (Figure 4b–d).

Figure 4 BDS-hEA inhibits the growth of HCC by inducing autophagy in tumor tissues. (a) Tumor growth status was real-time-monitored using a small animal live imaging system. (b) Tumor anatomy of mice. (c) Tumor volume statistical chart. (d) Tumor weight statistical chart. (e) Beclin-1, p62, and LC3-II expression levels were determined by immunohistochemical analysis. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control group. # p < 0.05 and ## p < 0.01 vs BDS-hEA group. ns means no significance vs BDS-hEA group.
Figure 4

BDS-hEA inhibits the growth of HCC by inducing autophagy in tumor tissues. (a) Tumor growth status was real-time-monitored using a small animal live imaging system. (b) Tumor anatomy of mice. (c) Tumor volume statistical chart. (d) Tumor weight statistical chart. (e) Beclin-1, p62, and LC3-II expression levels were determined by immunohistochemical analysis. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control group. # p < 0.05 and ## p < 0.01 vs BDS-hEA group. ns means no significance vs BDS-hEA group.

To further validate the induction of autophagy by BDS-hEA in tumor tissues, we conducted immunohistochemical analysis to assess the protein levels of p62, Beclin-1, and LC3-II. As shown in Figure 4e, the BDS-hEA group showed a significant increase in the expression levels of p62, Beclin-1, and LC3-II (yellow or brown particles appeared in the cytoplasm or cell membrane) when in comparison to the control group. When combined with CQ, there was a further upregulation observed in these proteins. This phenomenon possibly ascribes to the inhibitory effect of CQ on late-stage autophagy, which prevents the fusion between autophagosomes and lysosomes, thereby hindering the degradation process of these proteins. Altogether, these results indicate that activation of autophagy is responsible for the inhibitory effects of BDS-hEA on transplanted HCC tumors.

3.5 BDS-hEA inhibits tumor proliferation, migration, and angiogenesis by inducing autophagic apoptosis in tumor tissues

In order to investigate the potential mechanism of BDS-hEA inhibiting HCC tumor growth, immunofluorescence staining was employed to analyze the expression changes of tumor proliferation (ki67), migration (MMP-9), and angiogenesis (CD31 and VEGF). Figure 5a shows that BDS-hEA significantly downregulated the expression levels of ki67, MMP-9, CD31, and VEGF in comparison to the control group. However, it is noteworthy that the expression levels of these proteins were found to be significantly higher in the BDS-hEA + CQ group than those observed in the BDS-hEA group alone. The activation of autophagy was found to be concomitant with the apoptosis of tissue cells [14]. To further investigate their interrelationship, we assessed the extent of apoptosis in tumor tissues using TUNEL staining. As illustrated in Figure 5b, BDS-hEA significantly increased the population of red fluorescent apoptotic cells, whereas a decrease was noted in the BDS-hEA + CQ group when compared with the BDS-hEA alone. In all, the above results suggest that BDS-hEA exerts a tumor-suppressor role by inducing autophagy-dependent apoptosis to inhibit the expression of proteins associated with tumor proliferation, migration, and angiogenesis.

Figure 5 BDS-hEA inhibits tumor proliferation, migration, and angiogenesis by inducing autophagic apoptosis in tumor tissues. (a) Immunofluorescence staining was used to analyze the expression changes of ki67, MMP-9, CD31, and VEGF proteins. (b) Tumor tissue apoptosis was assessed using TUNEL staining. All results were repeated three times. Five fields were randomly selected for statistics. ***p < 0.001 vs control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 vs BDS-hEA group.
Figure 5

BDS-hEA inhibits tumor proliferation, migration, and angiogenesis by inducing autophagic apoptosis in tumor tissues. (a) Immunofluorescence staining was used to analyze the expression changes of ki67, MMP-9, CD31, and VEGF proteins. (b) Tumor tissue apoptosis was assessed using TUNEL staining. All results were repeated three times. Five fields were randomly selected for statistics. ***p < 0.001 vs control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 vs BDS-hEA group.

3.6 BDS-hEA induces autophagy in tumor tissues through modulation of the AMPK/AKT/mTOR signaling pathway

To gain deeper insights into the molecular regulatory mechanism of autophagy induced by BDS-hEA in vivo, the immunohistochemistry assay was conducted to assess the levels of p-AMPK, p-AKT, and p-mTOR in tumor tissues. The results indicated a significant rise in the rate of positive expression of p-AMPK, accompanied by a notable reduction in p-AKT and p-mTOR levels within the BDS-hEA group compared to the control group (Figure 6). However, when combined with CQ, changes in the expression of these proteins were not statistically significant compared to the BDS-hEA group, which may be due to the fact that CQ blocked the downstream autophagic flow but did not affect the occurrence of upstream autophagy. Collectively, our data indicate that BDS-hEA regulates autophagy in vivo by activating AMPK and inhibiting AKT/mTOR signaling.

Figure 6 BDS-hEA induces autophagy in tumor tissues through the modulation of the AMPK/AKT/mTOR signaling pathway. The immunohistochemistry assay was performed to assess the levels of p-AMPK, p-AKT, and p-mTOR in tumor tissues. All results were repeated three times. Five fields were randomly selected for statistics. **p < 0.01 and ***p < 0.001 vs control group; ns means no significance vs BDS-hEA group.
Figure 6

BDS-hEA induces autophagy in tumor tissues through the modulation of the AMPK/AKT/mTOR signaling pathway. The immunohistochemistry assay was performed to assess the levels of p-AMPK, p-AKT, and p-mTOR in tumor tissues. All results were repeated three times. Five fields were randomly selected for statistics. **p < 0.01 and ***p < 0.001 vs control group; ns means no significance vs BDS-hEA group.

4 Discussion

Angiogenesis is a multifaceted biological process involving the activation, proliferation, migration, and tube formation of endothelial cells, ultimately leading to the development of new blood vessels [15]. Neovascularization is crucial for the provision of essential nutrients and oxygen to tissues, thereby playing an indispensable role in facilitating tumor progression and metastasis [16]. Inhibition of angiogenesis has emerged as a promising therapeutic strategy against tumors. Endostatin is a 20 kDa C-terminal hydrolytic fragment derived from the XVIII collagen α 1-chain, which has been extensively studied as one of the endogenous angiogenesis inhibitors [17]. The protein drug Endostar, a recombinant form of human endostatin, received approval from China’s State Food and Drug Administration in 2005 for the treatment of non-small cell lung cancer [18], highlighting the potential efficacy of endostatin in cancer therapy. Angiostatin, the initial Kringle domain among four domains found in a 38 kDa internal proteolytic fragment of plasminogen, has been acknowledged as a potent endogenous inhibitor of angiogenesis and extensively demonstrated for its anti-tumor efficacy [19]. The primary obstacle hindering the future application of these drugs in clinical trials, however, lies in their limited therapeutic efficacy and short half-life [3]. In the past few years, tumor gene therapy has emerged as a valuable therapeutic strategy, complementing conventional treatments such as surgery, chemotherapy, and radiotherapy. Gene therapy offers distinct advantages such as sustained and localized expression of therapeutic gene products, low cost, and minimal side effects, making it a promising alternative to conventional protein drugs for cancer treatment [20]. However, the delivery efficiency and safety of gene therapy still need to be further improved.

The establishment of an efficient and secure delivery system is the foundation of gene therapy. Baculovirus, being an insect-specific virus, possesses several notable advantages over currently used viral vectors such as adenovirus, adeno-associated virus, retrovirus, and lentivirus. These advantages encompass its non-pathogenicity to humans, non-immunogenicity, ability to transduce both dividing and non-dividing cells, as well as lower the cell toxicity in mammalian cells [21,22]. Nevertheless, the application of baculovirus is constrained due to its susceptibility to complement system inactivation and the transient expression of exogenous genes [23]. In our previous work, we successfully developed a bivalent baculovirus gene delivery system by displaying the decay accelerating factor (DAF) on the viral surface to protect against complement attack while employing the Sleeping Beauty transposon system for efficient integration of exogenous genes into the host chromosome [5] and confirmed that the hybrid viruses had no significant inhibitory effect on the vitality and proliferation of vascular endothelial cells, indicating that our constructed viral vector is safe. We further utilized this system to express the fusion protein of human endothelin and angiostatin (BDS-hEA), confirming that the recombinant protein has significant inhibitory effects on tumor angiogenesis and growth in mouse HCC [5]. When combined with gemcitabine, it exhibited even stronger inhibition of tumor growth. Additionally, we found that BDS-hEA had less hepatotoxicity compared to gemcitabine, indicating that gene therapy mediated by baculovirus exhibits weaker toxic effects than chemotherapy drugs [6]. Given the excellent anti-tumor efficacy and safety of BDS-hEA, this study aims to further elucidate its anti-angiogenic mechanism.

Emerging evidence reveals a crucial role of autophagy in endothelial cells, the primary constituents of the vasculature responsible for delivering nutrients and oxygen to tissues. Furthermore, recent studies suggest that autophagy governs pathological angiogenesis, a characteristic feature of solid tumors [24]. The hyperproliferative nature of tumors readily lead to hypoxia and malnutrition within the tumor microenvironment. Under these adverse circumstances, heightened autophagy in blood vessels is emerging as a critical mechanism. Autophagy is a dynamic process encompassing the initiation, formation, maturation, and degradation of autophagosomes [25]. Multiple autophagy-related proteins actively participate in this intricate pathway. For instance, Beclin-1 plays a pivotal role in both the initiation and maturation stages of autophagosome biogenesis, while LC3-I undergoes transformation into LC3-II during the entire process of autophagosome formation [26]. Additionally, p62, an adaptor protein that negatively regulates autophagy, primarily participates in proteolytic degradation through the autophagy–lysosome system and the ubiquitin–proteasome system [27]. Therefore, increased levels of Beclin-1 and LC3-II, as well as decreased levels of p62, indicate the presence of autophagic activity. Our study observed an increased formation of autophagosomes, accompanied by upregulated expression of Beclin-1, LC3-II/LC3-I, and p62 (Figure 1a and d). Suppression of autophagy in vitro using 3-MA effectively reversed the aforementioned indicators (Figure 1b and e), indicating that BDS-hEA has the ability to trigger autophagy in vascular endothelial cells. Furthermore, the transplantation of mouse HCC further confirmed the capacity of BDS-hEA to trigger autophagy, as demonstrated by the upregulation of Beclin-1, LC3-II, and p62 proteins (Figure 4e). It is noteworthy that the activation of autophagy by BDS-hEA does not lead to a decrease in the level of p62, which serves as a marker for assessing the autophagic flux. When BDS-hEA is combined with the autophagy inhibitor CQ, there is a further increase in the levels of Beclin-1, LC3-II, and p62. This observation suggests that BDS-hEA impedes the autophagy flux, resulting in the accumulation of autophagosomes without subsequent degradation. Our findings suggest that BDS-hEA exerts effects similar to CQ, and further investigation is needed to determine whether it disrupts lysosomal membrane integrity and hinders fusion between autophagosomes and lysosomes. Recent research works have demonstrated that p62 can independently regulate oxidative stress and inflammation apart from its role in autophagy degradation [28]. Clinical investigations have revealed a correlation between the accumulation of p62 and unfavorable outcomes in patients diagnosed with HCC [29]. This may potentially explain the limited efficacy of anti-angiogenic drugs. Currently, we are exploring the relationship between the aberrant expression of p62 and the resistance to BDS-hEA treatment. Furthermore, ROS was found to be a key signaling molecule essential for regulating endothelial cell proliferation, migration, and angiogenesis while also playing an important role in mediating cellular autophagy [30]. Meng et al. found that fascaplysin can inhibit angiogenesis and increase ROS levels by activating autophagy, and blocking ROS production attenuates the fascaplysin-induced autophagic response [31]. Our findings provide additional evidence that BDS-hEA effectively enhanced ROS levels in a dose-dependent pattern (Figure 1c). Collectively, our results provide novel evidence that BDS-hEA has the ability to induce autophagy in vascular endothelial cells and HCC tumors.

The relationship between autophagy and angiogenesis remains inconclusive in various studies due to the autophagy’s dual nature. For instance, rapamycin-induced autophagy has been found to facilitate pro-angiogenic effects in the HUVECs [32]. Conversely, another study demonstrated that mebendazole induces autophagy in endothelial cells, leading to an anti-angiogenic effect [33]. Our investigation demonstrated that the functions of BDS-hEA-treated EA.hy926 cells were counteracted upon inhibition of autophagy, as evidenced by the reversal in cell viability, proliferation, invasion, migration, and angiogenesis as well as the expression levels of VEGF, VEGFR2, and CD31 (Figure 2). Furthermore, the tumor growth-suppressive properties of BDS-hEA in mice were compromised when autophagy was inhibited, as demonstrated by the results of Figure 4a–d. This inhibitory effect was ascribed to the promotion of apoptosis in tumor tissues and the restraint of proteins associated with tumor proliferation (ki67), metastasis (MMP-9), and angiogenesis (CD31 and VEGF) expression (Figure 5). Collectively, these findings underscore the role of autophagy as a suppressor in inhibiting HCC tumor growth mediated by BDS-hEA.

Studies have reported that the angiogenesis inhibitor bevacizumab can exacerbate hypoxia and nutritional deficiencies within the tumor microenvironment [34]. As a cellular sensor, mTOR exerts positive regulation on cellular nutrient uptake, energy metabolism, and stress response signaling [35]. Under stressful conditions such as nutrient deprivation and hypoxia, the AKT/mTOR axis is suppressed, thereby inducing autophagy and degradation of cellular components for energy acquisition [36]. Additionally, the AKT/mTOR pathway is also crucially involved in the regulation of neovascularization, and inhibiting its activity effectively suppresses angiogenesis [37]. For example, Wu et al. reported that the combination of Endostar and anti-PD-1 significantly inhibited lung cancer angiogenesis in mice by inhibiting the PI3K/AKT/mTOR signaling pathway to activate autophagy [18]. AMPK serves as another crucial intracellular sensor that plays a key role in negatively regulating cellular energy levels and inducing autophagy in response to conditions of oxidative stress and energy deprivation [38]. Upon stimuli, AMPK can activate autophagy by directly phosphorylating and activating ULK1 or indirectly activating ULK1 with TSC2-mediated mTOR inhibition [39]. Our results provided evidence supporting the induction of autophagy by BDS-hEA both in vitro (Figure 3) and in vivo (Figure 6), through the activation of AMPK and inhibition of AKT/mTOR signaling pathways.

5 Conclusions

In summary, our investigation revealed that BDS-hEA induces autophagy in vascular endothelial cells and HCC tumors by activating AMPK and suppressing AKT/mTOR signaling pathways and that autophagy inhibition blocks the BDS-hEA-induced functions of vascular endothelial cell proliferation, metastasis, and angiogenesis as well as HCC tumor growth. These findings provide a theoretical foundation for developing targeted molecules against the autophagy pathway to enhance the therapeutic efficacy of antiangiogenic drugs.

# Equal contribution to this work.

  1. Funding information: This work was supported by the Ningxia Natural Science Project (2023AAC03167), Outstanding Youth Support Project of Ningxia Education Department (NYG2022032), and General Hospital of Ningxia Medical University Project (YKDZY2022013).

  2. Author contributions: T.W. and J.C. completed the experiment together and wrote the original draft. Y.J. and X.C. participated in animal experiments. S.D. and Q.L. guided in vitro and in vivo experiments. Z.W. performed project administration, supervision, revised the final draft, and provided financial and technical support for the experiment. All authors read and approved the final manuscript.

  3. Conflict of interest: Authors state no conflict of interest.

  4. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2024-03-04
Revised: 2024-05-27
Accepted: 2024-06-04
Published Online: 2024-07-29

© 2024 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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  116. Electroacupuncture on GB acupoints improves osteoporosis via the estradiol–PI3K–Akt signaling pathway
  117. Renalase protects against podocyte injury by inhibiting oxidative stress and apoptosis in diabetic nephropathy
  118. Review: Dicranostigma leptopodum: A peculiar plant of Papaveraceae
  119. Combination effect of flavonoids attenuates lung cancer cell proliferation by inhibiting the STAT3 and FAK signaling pathway
  120. Renal microangiopathy and immune complex glomerulonephritis induced by anti-tumour agents: A case report
  121. Correlation analysis of AVPR1a and AVPR2 with abnormal water and sodium and potassium metabolism in rats
  122. Gastrointestinal health anti-diarrheal mixture relieves spleen deficiency-induced diarrhea through regulating gut microbiota
  123. Myriad factors and pathways influencing tumor radiotherapy resistance
  124. Exploring the effects of culture conditions on Yapsin (YPS) gene expression in Nakaseomyces glabratus
  125. Screening of prognostic core genes based on cell–cell interaction in the peripheral blood of patients with sepsis
  126. Coagulation factor II thrombin receptor as a promising biomarker in breast cancer management
  127. Ileocecal mucinous carcinoma misdiagnosed as incarcerated hernia: A case report
  128. Methyltransferase like 13 promotes malignant behaviors of bladder cancer cells through targeting PI3K/ATK signaling pathway
  129. The debate between electricity and heat, efficacy and safety of irreversible electroporation and radiofrequency ablation in the treatment of liver cancer: A meta-analysis
  130. ZAG promotes colorectal cancer cell proliferation and epithelial–mesenchymal transition by promoting lipid synthesis
  131. Baicalein inhibits NLRP3 inflammasome activation and mitigates placental inflammation and oxidative stress in gestational diabetes mellitus
  132. Impact of SWCNT-conjugated senna leaf extract on breast cancer cells: A potential apoptotic therapeutic strategy
  133. MFAP5 inhibits the malignant progression of endometrial cancer cells in vitro
  134. Major ozonated autohemotherapy promoted functional recovery following spinal cord injury in adult rats via the inhibition of oxidative stress and inflammation
  135. Axodendritic targeting of TAU and MAP2 and microtubule polarization in iPSC-derived versus SH-SY5Y-derived human neurons
  136. Differential expression of phosphoinositide 3-kinase/protein kinase B and Toll-like receptor/nuclear factor kappa B signaling pathways in experimental obesity Wistar rat model
  137. The therapeutic potential of targeting Oncostatin M and the interleukin-6 family in retinal diseases: A comprehensive review
  138. BA inhibits LPS-stimulated inflammatory response and apoptosis in human middle ear epithelial cells by regulating the Nf-Kb/Iκbα axis
  139. Role of circRMRP and circRPL27 in chronic obstructive pulmonary disease
  140. Investigating the role of hyperexpressed HCN1 in inducing myocardial infarction through activation of the NF-κB signaling pathway
  141. Characterization of phenolic compounds and evaluation of anti-diabetic potential in Cannabis sativa L. seeds: In vivo, in vitro, and in silico studies
  142. Quantitative immunohistochemistry analysis of breast Ki67 based on artificial intelligence
  143. Ecology and Environmental Science
  144. Screening of different growth conditions of Bacillus subtilis isolated from membrane-less microbial fuel cell toward antimicrobial activity profiling
  145. Degradation of a mixture of 13 polycyclic aromatic hydrocarbons by commercial effective microorganisms
  146. Evaluation of the impact of two citrus plants on the variation of Panonychus citri (Acari: Tetranychidae) and beneficial phytoseiid mites
  147. Prediction of present and future distribution areas of Juniperus drupacea Labill and determination of ethnobotany properties in Antalya Province, Türkiye
  148. Population genetics of Todarodes pacificus (Cephalopoda: Ommastrephidae) in the northwest Pacific Ocean via GBS sequencing
  149. A comparative analysis of dendrometric, macromorphological, and micromorphological characteristics of Pistacia atlantica subsp. atlantica and Pistacia terebinthus in the middle Atlas region of Morocco
  150. Macrofungal sporocarp community in the lichen Scots pine forests
  151. Assessing the proximate compositions of indigenous forage species in Yemen’s pastoral rangelands
  152. Food Science
  153. Gut microbiota changes associated with low-carbohydrate diet intervention for obesity
  154. Reexamination of Aspergillus cristatus phylogeny in dark tea: Characteristics of the mitochondrial genome
  155. Differences in the flavonoid composition of the leaves, fruits, and branches of mulberry are distinguished based on a plant metabolomics approach
  156. Investigating the impact of wet rendering (solventless method) on PUFA-rich oil from catfish (Clarias magur) viscera
  157. Non-linear associations between cardiovascular metabolic indices and metabolic-associated fatty liver disease: A cross-sectional study in the US population (2017–2020)
  158. Knockdown of USP7 alleviates atherosclerosis in ApoE-deficient mice by regulating EZH2 expression
  159. Utility of dairy microbiome as a tool for authentication and traceability
  160. Agriculture
  161. Enhancing faba bean (Vicia faba L.) productivity through establishing the area-specific fertilizer rate recommendation in southwest Ethiopia
  162. Impact of novel herbicide based on synthetic auxins and ALS inhibitor on weed control
  163. Perspectives of pteridophytes microbiome for bioremediation in agricultural applications
  164. Fertilizer application parameters for drip-irrigated peanut based on the fertilizer effect function established from a “3414” field trial
  165. Improving the productivity and profitability of maize (Zea mays L.) using optimum blended inorganic fertilization
  166. Application of leaf multispectral analyzer in comparison to hyperspectral device to assess the diversity of spectral reflectance indices in wheat genotypes
  167. Animal Sciences
  168. Knockdown of ANP32E inhibits colorectal cancer cell growth and glycolysis by regulating the AKT/mTOR pathway
  169. Development of a detection chip for major pathogenic drug-resistant genes and drug targets in bovine respiratory system diseases
  170. Exploration of the genetic influence of MYOT and MB genes on the plumage coloration of Muscovy ducks
  171. Transcriptome analysis of adipose tissue in grazing cattle: Identifying key regulators of fat metabolism
  172. Comparison of nutritional value of the wild and cultivated spiny loaches at three growth stages
  173. Transcriptomic analysis of liver immune response in Chinese spiny frog (Quasipaa spinosa) infected with Proteus mirabilis
  174. Disruption of BCAA degradation is a critical characteristic of diabetic cardiomyopathy revealed by integrated transcriptome and metabolome analysis
  175. Plant Sciences
  176. Effect of long-term in-row branch covering on soil microorganisms in pear orchards
  177. Photosynthetic physiological characteristics, growth performance, and element concentrations reveal the calcicole–calcifuge behaviors of three Camellia species
  178. Transcriptome analysis reveals the mechanism of NaHCO3 promoting tobacco leaf maturation
  179. Bioinformatics, expression analysis, and functional verification of allene oxide synthase gene HvnAOS1 and HvnAOS2 in qingke
  180. Water, nitrogen, and phosphorus coupling improves gray jujube fruit quality and yield
  181. Improving grape fruit quality through soil conditioner: Insights from RNA-seq analysis of Cabernet Sauvignon roots
  182. Role of Embinin in the reabsorption of nucleus pulposus in lumbar disc herniation: Promotion of nucleus pulposus neovascularization and apoptosis of nucleus pulposus cells
  183. Revealing the effects of amino acid, organic acid, and phytohormones on the germination of tomato seeds under salinity stress
  184. Combined effects of nitrogen fertilizer and biochar on the growth, yield, and quality of pepper
  185. Comprehensive phytochemical and toxicological analysis of Chenopodium ambrosioides (L.) fractions
  186. Impact of “3414” fertilization on the yield and quality of greenhouse tomatoes
  187. Exploring the coupling mode of water and fertilizer for improving growth, fruit quality, and yield of the pear in the arid region
  188. Metagenomic analysis of endophytic bacteria in seed potato (Solanum tuberosum)
  189. Antibacterial, antifungal, and phytochemical properties of Salsola kali ethanolic extract
  190. Exploring the hepatoprotective properties of citronellol: In vitro and in silico studies on ethanol-induced damage in HepG2 cells
  191. Enhanced osmotic dehydration of watermelon rind using honey–sucrose solutions: A study on pre-treatment efficacy and mass transfer kinetics
  192. Effects of exogenous 2,4-epibrassinolide on photosynthetic traits of 53 cowpea varieties under NaCl stress
  193. Comparative transcriptome analysis of maize (Zea mays L.) seedlings in response to copper stress
  194. An optimization method for measuring the stomata in cassava (Manihot esculenta Crantz) under multiple abiotic stresses
  195. Fosinopril inhibits Ang II-induced VSMC proliferation, phenotype transformation, migration, and oxidative stress through the TGF-β1/Smad signaling pathway
  196. Antioxidant and antimicrobial activities of Salsola imbricata methanolic extract and its phytochemical characterization
  197. Bioengineering and Biotechnology
  198. Absorbable calcium and phosphorus bioactive membranes promote bone marrow mesenchymal stem cells osteogenic differentiation for bone regeneration
  199. New advances in protein engineering for industrial applications: Key takeaways
  200. An overview of the production and use of Bacillus thuringiensis toxin
  201. Research progress of nanoparticles in diagnosis and treatment of hepatocellular carcinoma
  202. Bioelectrochemical biosensors for water quality assessment and wastewater monitoring
  203. PEI/MMNs@LNA-542 nanoparticles alleviate ICU-acquired weakness through targeted autophagy inhibition and mitochondrial protection
  204. Unleashing of cytotoxic effects of thymoquinone-bovine serum albumin nanoparticles on A549 lung cancer cells
  205. Erratum
  206. Erratum to “Investigating the association between dietary patterns and glycemic control among children and adolescents with T1DM”
  207. Erratum to “Activation of hypermethylated P2RY1 mitigates gastric cancer by promoting apoptosis and inhibiting proliferation”
  208. Retraction
  209. Retraction to “MiR-223-3p regulates cell viability, migration, invasion, and apoptosis of non-small cell lung cancer cells by targeting RHOB”
  210. Retraction to “A data mining technique for detecting malignant mesothelioma cancer using multiple regression analysis”
  211. Special Issue on Advances in Neurodegenerative Disease Research and Treatment
  212. Transplantation of human neural stem cell prevents symptomatic motor behavior disability in a rat model of Parkinson’s disease
  213. Special Issue on Multi-omics
  214. Inflammasome complex genes with clinical relevance suggest potential as therapeutic targets for anti-tumor drugs in clear cell renal cell carcinoma
  215. Gastroesophageal varices in primary biliary cholangitis with anti-centromere antibody positivity: Early onset?
https://doi.org/10.1515/biol-2022-0914
Keywords for this article
hepatocellular carcinoma; baculovirus; endostatin-angiostatin; anti-angiogenesis; autophagy
Creative Commons
BY 4.0

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  125. Screening of prognostic core genes based on cell–cell interaction in the peripheral blood of patients with sepsis
  126. Coagulation factor II thrombin receptor as a promising biomarker in breast cancer management
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  128. Methyltransferase like 13 promotes malignant behaviors of bladder cancer cells through targeting PI3K/ATK signaling pathway
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  130. ZAG promotes colorectal cancer cell proliferation and epithelial–mesenchymal transition by promoting lipid synthesis
  131. Baicalein inhibits NLRP3 inflammasome activation and mitigates placental inflammation and oxidative stress in gestational diabetes mellitus
  132. Impact of SWCNT-conjugated senna leaf extract on breast cancer cells: A potential apoptotic therapeutic strategy
  133. MFAP5 inhibits the malignant progression of endometrial cancer cells in vitro
  134. Major ozonated autohemotherapy promoted functional recovery following spinal cord injury in adult rats via the inhibition of oxidative stress and inflammation
  135. Axodendritic targeting of TAU and MAP2 and microtubule polarization in iPSC-derived versus SH-SY5Y-derived human neurons
  136. Differential expression of phosphoinositide 3-kinase/protein kinase B and Toll-like receptor/nuclear factor kappa B signaling pathways in experimental obesity Wistar rat model
  137. The therapeutic potential of targeting Oncostatin M and the interleukin-6 family in retinal diseases: A comprehensive review
  138. BA inhibits LPS-stimulated inflammatory response and apoptosis in human middle ear epithelial cells by regulating the Nf-Kb/Iκbα axis
  139. Role of circRMRP and circRPL27 in chronic obstructive pulmonary disease
  140. Investigating the role of hyperexpressed HCN1 in inducing myocardial infarction through activation of the NF-κB signaling pathway
  141. Characterization of phenolic compounds and evaluation of anti-diabetic potential in Cannabis sativa L. seeds: In vivo, in vitro, and in silico studies
  142. Quantitative immunohistochemistry analysis of breast Ki67 based on artificial intelligence
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  145. Degradation of a mixture of 13 polycyclic aromatic hydrocarbons by commercial effective microorganisms
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  148. Population genetics of Todarodes pacificus (Cephalopoda: Ommastrephidae) in the northwest Pacific Ocean via GBS sequencing
  149. A comparative analysis of dendrometric, macromorphological, and micromorphological characteristics of Pistacia atlantica subsp. atlantica and Pistacia terebinthus in the middle Atlas region of Morocco
  150. Macrofungal sporocarp community in the lichen Scots pine forests
  151. Assessing the proximate compositions of indigenous forage species in Yemen’s pastoral rangelands
  152. Food Science
  153. Gut microbiota changes associated with low-carbohydrate diet intervention for obesity
  154. Reexamination of Aspergillus cristatus phylogeny in dark tea: Characteristics of the mitochondrial genome
  155. Differences in the flavonoid composition of the leaves, fruits, and branches of mulberry are distinguished based on a plant metabolomics approach
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  157. Non-linear associations between cardiovascular metabolic indices and metabolic-associated fatty liver disease: A cross-sectional study in the US population (2017–2020)
  158. Knockdown of USP7 alleviates atherosclerosis in ApoE-deficient mice by regulating EZH2 expression
  159. Utility of dairy microbiome as a tool for authentication and traceability
  160. Agriculture
  161. Enhancing faba bean (Vicia faba L.) productivity through establishing the area-specific fertilizer rate recommendation in southwest Ethiopia
  162. Impact of novel herbicide based on synthetic auxins and ALS inhibitor on weed control
  163. Perspectives of pteridophytes microbiome for bioremediation in agricultural applications
  164. Fertilizer application parameters for drip-irrigated peanut based on the fertilizer effect function established from a “3414” field trial
  165. Improving the productivity and profitability of maize (Zea mays L.) using optimum blended inorganic fertilization
  166. Application of leaf multispectral analyzer in comparison to hyperspectral device to assess the diversity of spectral reflectance indices in wheat genotypes
  167. Animal Sciences
  168. Knockdown of ANP32E inhibits colorectal cancer cell growth and glycolysis by regulating the AKT/mTOR pathway
  169. Development of a detection chip for major pathogenic drug-resistant genes and drug targets in bovine respiratory system diseases
  170. Exploration of the genetic influence of MYOT and MB genes on the plumage coloration of Muscovy ducks
  171. Transcriptome analysis of adipose tissue in grazing cattle: Identifying key regulators of fat metabolism
  172. Comparison of nutritional value of the wild and cultivated spiny loaches at three growth stages
  173. Transcriptomic analysis of liver immune response in Chinese spiny frog (Quasipaa spinosa) infected with Proteus mirabilis
  174. Disruption of BCAA degradation is a critical characteristic of diabetic cardiomyopathy revealed by integrated transcriptome and metabolome analysis
  175. Plant Sciences
  176. Effect of long-term in-row branch covering on soil microorganisms in pear orchards
  177. Photosynthetic physiological characteristics, growth performance, and element concentrations reveal the calcicole–calcifuge behaviors of three Camellia species
  178. Transcriptome analysis reveals the mechanism of NaHCO3 promoting tobacco leaf maturation
  179. Bioinformatics, expression analysis, and functional verification of allene oxide synthase gene HvnAOS1 and HvnAOS2 in qingke
  180. Water, nitrogen, and phosphorus coupling improves gray jujube fruit quality and yield
  181. Improving grape fruit quality through soil conditioner: Insights from RNA-seq analysis of Cabernet Sauvignon roots
  182. Role of Embinin in the reabsorption of nucleus pulposus in lumbar disc herniation: Promotion of nucleus pulposus neovascularization and apoptosis of nucleus pulposus cells
  183. Revealing the effects of amino acid, organic acid, and phytohormones on the germination of tomato seeds under salinity stress
  184. Combined effects of nitrogen fertilizer and biochar on the growth, yield, and quality of pepper
  185. Comprehensive phytochemical and toxicological analysis of Chenopodium ambrosioides (L.) fractions
  186. Impact of “3414” fertilization on the yield and quality of greenhouse tomatoes
  187. Exploring the coupling mode of water and fertilizer for improving growth, fruit quality, and yield of the pear in the arid region
  188. Metagenomic analysis of endophytic bacteria in seed potato (Solanum tuberosum)
  189. Antibacterial, antifungal, and phytochemical properties of Salsola kali ethanolic extract
  190. Exploring the hepatoprotective properties of citronellol: In vitro and in silico studies on ethanol-induced damage in HepG2 cells
  191. Enhanced osmotic dehydration of watermelon rind using honey–sucrose solutions: A study on pre-treatment efficacy and mass transfer kinetics
  192. Effects of exogenous 2,4-epibrassinolide on photosynthetic traits of 53 cowpea varieties under NaCl stress
  193. Comparative transcriptome analysis of maize (Zea mays L.) seedlings in response to copper stress
  194. An optimization method for measuring the stomata in cassava (Manihot esculenta Crantz) under multiple abiotic stresses
  195. Fosinopril inhibits Ang II-induced VSMC proliferation, phenotype transformation, migration, and oxidative stress through the TGF-β1/Smad signaling pathway
  196. Antioxidant and antimicrobial activities of Salsola imbricata methanolic extract and its phytochemical characterization
  197. Bioengineering and Biotechnology
  198. Absorbable calcium and phosphorus bioactive membranes promote bone marrow mesenchymal stem cells osteogenic differentiation for bone regeneration
  199. New advances in protein engineering for industrial applications: Key takeaways
  200. An overview of the production and use of Bacillus thuringiensis toxin
  201. Research progress of nanoparticles in diagnosis and treatment of hepatocellular carcinoma
  202. Bioelectrochemical biosensors for water quality assessment and wastewater monitoring
  203. PEI/MMNs@LNA-542 nanoparticles alleviate ICU-acquired weakness through targeted autophagy inhibition and mitochondrial protection
  204. Unleashing of cytotoxic effects of thymoquinone-bovine serum albumin nanoparticles on A549 lung cancer cells
  205. Erratum
  206. Erratum to “Investigating the association between dietary patterns and glycemic control among children and adolescents with T1DM”
  207. Erratum to “Activation of hypermethylated P2RY1 mitigates gastric cancer by promoting apoptosis and inhibiting proliferation”
  208. Retraction
  209. Retraction to “MiR-223-3p regulates cell viability, migration, invasion, and apoptosis of non-small cell lung cancer cells by targeting RHOB”
  210. Retraction to “A data mining technique for detecting malignant mesothelioma cancer using multiple regression analysis”
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