The secondary metabolites of Bacillus subtilis strain Z15 Induce Apoptosis in Hepatocellular Carcinoma Cells

doi:10.21203/rs.3.rs-2330612/v1

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The secondary metabolites of Bacillus subtilis strain Z15 Induce Apoptosis in Hepatocellular Carcinoma Cells

https://doi.org/10.21203/rs.3.rs-2330612/v1

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published 31 Oct, 2023

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The lipopeptides produced by Bacillus subtilis have anti-cancer potential. We had previously identified a secondary metabolite of B. subtilis strain Z15 (BS-Z15), which has an operon that regulates lipopeptide synthesis, and also demonstrated that the fermentation products of this strain exerted antioxidant and pro-immune effects. The purpose of this study was to investigate in vitro and in vivo the anticancer effects of BS-Z15 secondary metabolites (BS-Z15 SMs) on hepatocellular carcinoma (HCC) cells. BS-Z15 SMs significantly inhibited H22 cell-derived murine xenograft tumor growth without any systemic toxicity. In addition, BS-Z15 SMs decreased the viability of H22 cells and BEL-7404 cells in vitro with respective IC₅₀ values of 33.83µg/mL and 27.26 µg/mL. Consistent with this, BS-Z15 SMs induced apoptosis and G0/G1 phase arrest in the BEL-7404 cells, and the mitochondrial membrane potential was also significantly reduced in a dose-dependent manner. Mechanistically, BS-Z15 SMs upregulated the pro-apoptotic p53, Bax, cytochrome C and cleaved-caspase-3/9 proteins, and downregulated the anti-apoptotic Bcl-2.These findings suggest that the induction of apoptosis in HCC cells by BS-Z15 SMs may be related to the mitochondrial pathway. Thus, the secondary metabolites of B. subtilis strain Z15 are promising to become new anti-cancer drugs for the clinical treatment of liver cancer.

Secondary metabolite of Bacillus subtilis strain Z15

apoptosis

mitochondrial-dependent pathway

Hepatocellular Carcinoma Cells

In 2020, primary liver cancer ranked sixth among cancers diagnosed worldwide. It also has a high incidence and mortality rate and is the third leading cause of common cancer-related deaths, with about 906,000 new cases and 830,000 deaths. Hepatocellular carcinomas (HCCs) and intrahepatic cholangiocarcinomas (ICCs) are the predominant liver cancers, followed by other rare pathological types [1]. HCC is in fact one of the most prevalent malignancies, and there is a poor prognosis and high mortality rate associated with it [2]. At present, liver transplantation (LT), surgical resection, and radiofrequency ablation are the most available treatment modalities for HCC patients in the initial phase, but are associated with high rates of post-therapeutic recurrence and metastasis [3–5]. Due to the insidious nature of the early stages of HCC, most patients are diagnosticated at an advanced stage and therefore cannot benefit from curative surgery [6]. Advanced HCC is usually treated with several tyrosine kinase inhibitors, such as sorafenib, regorafenib, and lenvatinib, which have already been approved [7, 8], although their clinical efficacy is limited due to high resistance and toxicity [9]. As a result, it is essential to develop drugs with a high efficacy but low toxicity for treating advanced HCC.

In recent years, a variety of bioactive molecules derived from microbial metabolites have emerged as a potential sources for research into new drugs. The non-pathogenic bacterium B.subtilis is commonly found in the environment, and produces high levels of lipopeptides such as surfactin, fengycin and iturin[10].These lipopeptides have amphiphilic structures, including hydrophilic cyclic peptides and hydrophobic fatty acid chains[11]. Lipopeptides have documented anti-fungal[12], anti-inflammatory[13], anti-bacterial[14], immunomodulatory[15] and anti-tumor ^[16]effects, and exhibit good biosafety. Several previous studies have suggested that B. subtilis lipopeptides can induce apoptosis in cancer cells, arrest the cell cycle and inhibit cancer cell invasion. Specifically, surfactin inhibited the multiplication rate of the LoVo colorectal cancer cell line through blocking the cell cycle and inducing apoptosis[16], iturinA disrupted the cell cycle, and induced apoptosis and autophagy through ROS-dependent manner in human HCC cells[17],and fengycin arrested the cell cycle in human lung cancer 95D cells through downregulation of cyclin D1 and cyclin dependent kinase 4 (CDK4), as well as inducing apoptosis[18].

We had previously isolated the B. subtilis strain Z15 (BS-Z15) from soil, and identified an operon by genomic analysis that regulates the synthesis of various lipopeptides such as fengycin, mycosubtilin and surfactin[19]. Zhao et al. found that intragastrically administered BS-Z15 secondary metabolites did not have overt toxic side effects on the main organs in mice[20]. Furthermore, Chen et al. found that these secondary metabolites increased superoxide dismutase activity in the liver of mice, dramatically reduced MDA levels, and prevented body weight gain[21]. Yang et al. also demonstrated that BS-Z15 secondary metabolites can affect body weight gain in mouse via changing the composition of the gut microbiota[22]. However, the anti-cancer activity of BS-Z15 secondary metabolites and the mechanisms that underlie it remain unknown. In this study, we prepared acetone extract of B. subtilis strain Z15 secondary metabolites (BS-Z15 SMs) and analyzed their effects on hepatoma growth. As a result, BS-Z15 SMs effectively inhibited both murine (H22) and human (BEL-7404) hepatoma cells proliferation in vitro.The main mechanism is to arrested cells in the G0/G1 phase and induced apoptosis in a mitochondria-dependent manner.In addition,BS-Z15 SMs stalled the growth of H22-derived xenografts in mice without any toxic effects.Therefore, BS-Z15 SMs is a safe and effective drug for the treatment of liver cancer, and warrants further studies.

2.1. Preparation of BS-Z15 SMs

B. Subtilis strain Z15 secondary metabolites were derived as described by Zhao et al[23]. Briefly, a single colony of B. Subtilis strain Z15 was picked and inoculated in beef paste peptone liquid medium. After incubating at 37℃ and 180 rpm for 18h, the broth was centrifuged at 12000 rpm for 20 min, and the clarified supernatant was aspirated. The proteins in the supernatant were precipitated with HCl (pH adjusted to 2) at 4°C, and centrifuged at 12000 rpm for 20 min. The precipitate was resuspended in sterile water, and chilled acetone was added for further extraction. We mixed the solution shaking vigorously and allowed it to stand at 4°C.Once the layers were delaminated, the upper organic phase was aspirated, and the residual acetone was removed with a rotary evaporator. This acetone extract of B. Subtilis-Z15 secondary metabolites (referred to as BS-Z15 SMs) was fractionated and stored at -20°C, and filtered through a 0.22µm sterile filter prior to the experiments.

2.2. Cell Culture

The H22 and BEL-7404 cell lines were provided by the Xinjiang Key Laboratory of Special Species Conservation and Regulatory Biology, Xinjiang Normal University (Urumqi, Xinjiang, China). The cells were cultured in RPMI-1640 medium containing 1% penicillin-streptomycin solution and 10% FBS at 37°C under 5% CO₂.

2.3. Experimental animals

Thirty-two male Kunming mice (6–8 weeks old) were purchased from Animal Experiment Center, Xinjiang Medical University (Urumqi, Xinjiang, China).The experimental mice were fed in a standard animal housing at Xinjiang Normal University, which is strictly controlled by temperature and light and free of specific pathogens. All animal experiments were followed the guidelines of the Animal Experimentation Center of Xinjiang Medical University.

2.4. Tumor induction

A subcutaneous graft tumor model of HCC was established using H22 cells. Briefly, 1×10⁶ H22 cells suspended in 100 µL PBS were subcutaneously injected into the right axilla of Kunming mice. 6 days later, the mice were randomly distributed into 4 groups of 8 mice each. The control group was injected intraperitoneally with 200 µL sterile saline each day, while the positive control group received 2 mg/kg cisplatin once every two days. The experimental groups received 30 mg/kg or 100 mg/kg BS-Z15 SMs intraperitoneally every day.The tumor dimensions were measured with a Vernier caliper every two days, and the mice were weighed. The tumor volume (mm³) was calculated as (length×width²)/2. The mice were euthanized after 15 days of treatment, and the tumors and main organs were harvested. Then weighed these tissue samples and placed in 4% formaldehyde for fixation.

2.5. H&E staining

The fixed tissues were dehydrated through an ethanol gradient, immersed in paraffin for embedding, and the paraffin blocks were cut into 5µm-thick sections when the paraffin was completely solidified, which were then stained with hematoxylin for 10 min and split with 1% hydrochloric acid ethanol for 10 s. The stained sections were viewed under a microscope and analyzed using an image analysis software.

2.6. MTT assay

H22 and BEL-7404 cells at the logarithmic growth stage were resuspended in culture medium and inoculated in 96-well culture plates at a density of 5000 cells/well and incubated overnight to adhere to the wall. The H22 cells were treated with 0, 10, 20, 40 and 80 µg/mL BS-Z15 SMs for 24 h, and BEL-7404 cells were treated with 0, 15, 25, 35 and 45 µg/mL of the drug for 24, 48 or 72h. At the same time, the positive control group was treated with 20 µg/ml cisplatin. At the respective time-points, 0.1 mg/mL MTT was added to the respective well, and the cells were incubated for another 4h. After dissolving the formazan crystals with 100 µL DMSO per well, the optical density(OD) values of each well were measured at 490 nm using a microplate reader (Benchmark Plus, Bio-Rad, USA). The viability (%) was calculated as (OD _treated/OD _control) ×100%. The experiment was repeated thrice.

2.7. Hoechst 33258 staining

BEL-7404 cells were grown in 6-well culture plates at a density of 1×10⁵ cells/well and cultured overnight. After attachment, cells were treated with BS-Z15 SMs (0, 15, 25, and 35 µg/mL) for 24 h, the medium was removed, and then washed cells twice with PBS. Finally, fixed cells for 10 minutes at 4℃ with chilled 4% paraformaldehyde, then washed twice with PBS. The cells were stained with 500 µL Hoechst 33258 staining solution for 10 min in the dark, and observed with an inverted fluorescence microscope (Eclipse E20, Nikon, Japan).

2.8. Cell cycle analysis

BEL-7404 cells were planted in 60 mm sterile culture dishes and cultured for 24 hours. The adherent cells were treated with BS-Z15 SMs (0, 15, 25 and 35 µg/mL), or 20 µg/mL cisplatin, for 24 h. The suitably treated cells were washed twice with cold PBS, fixed overnight in ice-cold 70% ethanol at 4℃, and then added the 400 µL propidium iodide (PI) and 100 µL RNase A staining solution and warm incubation for 30 min at 37℃ protected from light.The cell cycle distribution was analyzed by flow cytometry (FACS Calibur, BD Biosciences, USA). The experiment was repeated thrice.

2.9. Apoptosis assay

Apoptosis was detected using the Annexin V-FITC/PI Apoptosis Detection Kit as directed by the manufacturer. We seeded 4×10⁵ BEL-7404 cells in each 60 mm cell culture dish and cultured for 24 h.Following treatment with 0 to 35 µg/mL BS-Z15 SMs or 20 µg/mL cisplatin for 24 h, the adherent cells were harvested and washed twice with cold PBS. After resuspending the cells with 500 µL binding buffer, they were stained with 5 µL Annexin V-FITC and 5 µL PI for 15 min in a dark environment. The apoptotic cells were detected by flow cytometry (FACS Calibur, BD Biosciences, USA).

2.10. Detection of mitochondrial membrane potential

BEL-7404 cells were seeded into a 6-well plate at the density of 2×10⁵ cells/well and cultured for 24 h. After treating with BS-Z15 SMs (0, 15, 25 and 35 µg/mL) for 24 h, the cells were resuspended in 500 µL culture medium and 500 µL JC-1 staining solution. The cells were incubated for 20–30 min at 37°C, and observed under a fluorescence microscope (Eclipse E20, Nikon, Japan) at the emission wavelength of 540 nm.

2.11. Western blotting

The suitably treated BEL-7404 were harvested and lysed with RIPA buffer on ice for 30 min, then centrifuged, and the supernatant was aspirated. The amount of total protein in the cells was measured using a BCA protein assay kit. Equal amounts of proteins from per sample were separated electrophoretically on a 12% SDS-PA gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. After the transfer was completed, the PVDF membranes were placed in 5% skim milk and blocked by slow shaking on a shaker at room temperature for 2 h. Then the blots were incubated overnight with primary antibodies at 4°C, washed several times with TBST buffer, and probed with the horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1.5 h. The protein bands were detected using the ECL assay kit and Image J software was used to analyze their optical density, with β-actin as the internal control. The experiment was repeated thrice.

2.12. Statistical analysis

All experimental data are expressed as the mean ± standard deviation (SD) of three independent replicate experiments. One-way ANOVA was used for data analysis. GraphPad Prism 9.0 was used for statistical analysis. It was considered statistically significant when P < 0.05.

3.1. BS-Z15 SMs inhibited the tumor growth in vivo

We established H22-derived subcutaneous xenografts in Kunming mice to confirm the inhibition of tumor growth by BS-Z15 SMs in vivo. After 6 days following the injection of H22 cells, various doses of BS-Z15 SMs or cisplatin were administered to tumor-bearing mice. Compared to untreated controls, BS-Z15 SMs (30 mg/kg or 100 mg/kg) and cisplatin significantly inhibited tumor growth. (Fig. 1A-C). Unlike cisplatin, however, BS-Z15 SMs did not result in any significant weight loss in the mice (Fig. 1D). Furthermore, the liver tissues of mice treated with BS-Z15 SMs had intact lobular structures and regular cytoarchitecture, whereas cisplatin treatment led to significant pathological changes in the liver. No obvious signs of toxicity were observed in the spleen and kidney tissues in any of the treatment groups (Fig. 1E). Thus, BS-Z15 SMs exhibited potent anti-tumor activity with low toxicity and are a better alternative to cisplatin for developing novel drugs against HCC.

3.2. BS-Z15 SMs inhibited the growth of H22 and BEL-7404 cells in vitro

We treated H22 and BEL-7404 cells with BS-Z15 SMs and measured the viability of the cells through microscopic examination and the MTT assay. We observed significant morphological changes in H22 and BEL-7404 cells treated with various doses of BS-Z15 SMs compared to the untreated control group under an inverted microscope, and the drug-treated cells gradually shrank and became rounded (Fig. 2A).The viability rates of the two cancer cell lines after BS-Z15 SMs treatment were measured by MTT assay (Fig. 2B).BS-Z15 SMs also dramatically reduced the the percentage of viable H22 and BEL-7404 cells in a time- and dose-dependent trend (Fig. 2B), with respective IC₅₀ values of 33.83 µg/mL and 27.26 µg/mL at 24 h.

3.3. BS-Z15 SMs arrested the cell cycle of BEL-7404 cells

We next analyzed whether the viable cells remaining after BS-Z15 SMs treatment grow normally or are blocked during cell cycle transition. BEL-7404 cells treated with BS-Z15 SMs for 24 h will significantly increase the proportion of cells in G0/G1 and sub-G1 phases while decreasing the proportion of cells in S phase, suggesting that BS-Z15 SMs block the cell cycle in G0/G1 phase(Fig. 3).

3.4. BS-Z15 SMs induced apoptosis in BEL-7404 Cells

BEL-7404 cells were treated with BS-Z15 SMs for 24 h, and apoptosis was measured by detecting apoptotic changes in nuclei using Hoechst 33258 staining. In the control group, the nuclei were homogeneously stained, while cells treated with BS-Z15 SMs revealed, bright, condensed chromatin and nuclear fragmentation, which were indicative of apoptosis and this effect was more pronounced with increasing concentrations of BS-Z15 SMs (Fig. 4A). Annexin V-FITC/PI double-staining assay was used to further determine the apoptosis rates of cells in different treatment groups. As shown in Fig. 4B, BS-Z15 SMs increased apoptosis dose-dependently in BEL-7404 cells. Treatment with 25 µg/mL BS-Z15 SMs increased the apoptosis rates to 60.17 ± 1.29% compared to only 9.27 ± 1.46% in the untreated controls, and 44.73 ± 0.96% in the cisplatin-treated group. In addition, in comparison with the control group, there was a significant increase in necrosis (0.87%) following treatment with 35 µg/mL of BS-Z15 SMs, albeit not in a concentration-dependent way (Fig. 4B). Consistent with the above findings, BS-Z15 SMs also increased the expression of pro-apoptotic proteins Bax and p53, and decreased the expression of anti-apoptotic protein Bcl-2 in a concentration-dependent manner (Fig. 4C). Taken together, BS-Z15 SMs inhibited the growth of hepatoma cells via inducing apoptosis.

3.5. BS-Z15 SMs reduced mitochondrial membrane potential (∆ψm)

A characteristic feature of the early phases of apoptosis is the decrease in mitochondrial membrane potential. Several Bcl-2 family of proteins play an important role in mitochondrial membrane integrity, including the pro-apoptotic Bax and anti-apoptotic Bcl-2[24]. We measured the mitochondrial membrane potential (Δψm) in BEL-7404 cells using the JC-1 dye, which aggregates and shows red fluorescence when the Δψm is high, and remains a monomer and shows green fluorescence at lower Δψm[25]. As shown in Fig. 5A, the untreated cells emitted red fluorescence, while BS-Z15 SMs treatment significantly increased the proportion of green fluorescent cells, and decreased the red/green fluorescence ratio in a concentration-dependent manner(Fig. 5A). Thus, BS-Z15 SMs treatment reduced Δψm in the BEL-7404 cells, thereby disrupting the integrity of the mitochondrial membrane. The results of the western blotting assay were consistent with this, cytochrome C levels increased after treatment with BS-Z15 SMs, which was accompanied by a decrease in the levels of the downstream factors caspase-9 and caspase-3, and upregulation of the cleaved caspases-3 and cleaved caspases-9. In addition, BS-Z15 SMs downregulated the levels of cleaved caspase-8(Fig. 5B). Since caspase-8 activation is a marker of the extrinsic apoptosis pathway, these findings indicate that BS-Z15 SMs induced apoptosis in the BEL-7404 cells via the intrinsic mitochondrial pathway.

Chemotherapeutic drugs are beset with problems such as high costs, toxic side effects, and the drug tolerance, which warrants an urgent need to find potential alternatives. Natural compounds derived from microorganisms and plants are increasingly being employed for developing novel anti-cancer drugs on account of their high bioactivity and low toxicity[26]. Lipopeptides extracted from B. subtilis have exhibited significant anti-cancer activity along with good biosafety [11].

In previous studies, we had isolated the B. subtilis strain Z15 from soil, and identified an operon that regulates synthesis of lipopeptides, such as fengycin, mycosubtilin and surfactin[19]. Previous studies reported that surfactin was effective against human breast cancer [27] and cervical cancer [28] cells. Iturin is also cytotoxic to colorectal adenocarcinoma and alveolar adenocarcinoma cells [29], whereas fengycin can inhibit the proliferation of colon cancer [30] and lung cancer cells [18]. In this study, we have shown for the first time that the acetone extract of B. subtilis strain Z15 secondary metabolites inhibited the growth of murine and human hepatoma cells in vitro and in vivo. Furthermore, BS-Z15 SMs markedly inhibited the growth of H22 xenografts to a similar extent as cisplatin, without the systemic and hepatic toxicity of the BS-Z15 SMs.

Several chemotherapeutic targets inhibit tumor progression via arresting the cell cycle and inducing apoptosis [31]. We found that BS-Z15 SMs blocked the cell cycle transition at the G0/G1 phase and also increased the percentage of apoptotic cells. Surfactin from B.subtilis has previously been shown to induce cell cycle arrest in LoVo cells at the G0/G1 phase[32]. The iturin A-like lipopeptide also blocked the transition of Caco-2 cells at the G1 phase [33]. Thus, the anti-proliferative functions of B. subtilis lipopeptides can be attributed to their ability to arrest the cell cycle.

In biology, apoptosis is one of the programmed death of cells[34], and the predominant mechanism of tumor cell killing by many anti-tumor drugs. A majority of studies on the antitumor effects of lipopeptides produced by B. subtilis have fastened on apoptosis induction [35]. Surfactin derived from B. subtilis induces apoptosis in human oral squamous cell carcinoma via a ROS-regulated mitochondrial pathway[36]. Iturin A induces apoptosis in HepG2 cells by disrupting mitochondrial integrity and releasing cytochrome c [37]. Cells normally have a high mitochondrial membrane potential, which decreases in response to stressful stimuli that enhance the permeability of mitochondrial membrane[24]. The intrinsic mitochondrial apoptosis pathway is controlled by Bcl-2 and Bax[38, 39]. Bax triggers the cytochrome c release into the cytoplasm by increasing the permeability of the mitochondrial membrane, which then induces downstream activation of caspase-9 and caspase-3 and ultimately activates the intrinsic apoptotic pathway [40]. In this study, BS-Z15 SMs decreased the mitochondrial membrane potential of BEL-7404 cells and upregulated Bax, which corresponded to increased levels of cytochrome c and the cleaved caspases. In addition, BS-Z15 SMs inhibited the cleavage of caspase-8, which is activated and plays a key role in the exogenous apoptotic pathway [41]. These findings indicate that BS-Z15 SMs plays its anti-tumor effects by inducing apoptosis via the intrinsic mitochondrial pathway. P53 is a tumor suppressor gene[42] that arrests the cell cycle and induces senescence, and promotes apoptosis via extrinsic death receptor-mediated and intrinsic mitochondrial pathways in case the DNA damage cannot be repaired[43]. Consistent with the pro-apoptotic effects of BS-Z15 SMs, p53 protein expression was upregulated in cells treated with BS-Z15 SMs.

In conclusion, B. subtilis strain Z15 metabolites inhibited the proliferation of hepatoma cells by arresting cell cycle and inducing apoptosis through the mitochondrial pathway. Furthermore, BS-Z15 SMs inhibited H22 tumor growth in vivo without any significant side effects. Therefore, B. subtilis strain Z15 metabolites may be used to develop safe and effective drugs for treating liver cancer.

Given the technical challenges of isolating pure lipopeptides form the fermentation broth of B. subtilis, and the resulting high costs, mixture of lipopeptide homologs are now widely used to evaluate their anti-tumor activity [35, 44, 45].The lipopeptides present in the metabolites of B. subtilis strain Z15 have similar molecular weights and structures. Therefore, the actual lipopeptides with anti-cancer potential and their structures need to be explored further. This work is currently underway in our laboratory.

Author Contributions: All authors contributed to the study conception and design. Conceptualization :Huixin Zhao, Heping Zhao and Jinyu Li ; Methodology: Xiyuan Cao , Hui Zhang , Jia You and Weiquan Wang ; Formal analysis and Validation: Qi Zhang , Li Yin, Qinshuang Mei and Xiaorong Zhang ; Investigation and data curation: Reyihanguli Aimaier , Haoran Li ,Wenzhi Cao; Writing—original draft preparation: Reyihanguli Aimaier , Haoran Li ,Wenzhi Cao; Writing—review and editing: Huixin Zhao and Jinyu Li; Supervision:Huixin Zhao, Heping Zhao and Jinyu Li; Project administration: Huixin Zhao, Heping Zhao. All authors have read and agreed to the published version of the manuscript.

Funding: This work was supported by grants from Scientific Research Program of Colleges and Universities in Xinjiang (No. XJEDU2021I023) , Natural Science Foundation of China (No.32160074), and the Open Project of Key Laboratory in Xinjiang (No. 2020D4010).

Institutional Review Board Statement: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. Animal protocol approval: Xinjiang Medical University, SYXK (Xin) 2018–0003.

Data Availability Statement: The datasets supporting the conclusions of this article are included within the article and its additional file.Data will be made available on reasonable request. All authors ensure that all data and materials, as well as software applications or custom code, support their published claims and adhere to domain standards.

Conflicts of Interest: The authors declare no conflict of interest.

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published 31 Oct, 2023

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The secondary metabolites of Bacillus subtilis strain Z15 Induce Apoptosis in Hepatocellular Carcinoma Cells

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Preparation of BS-Z15 SMs

2.2. Cell Culture

2.3. Experimental animals

2.4. Tumor induction

2.5. H&E staining

2.6. MTT assay

2.7. Hoechst 33258 staining

2.8. Cell cycle analysis

2.9. Apoptosis assay

2.10. Detection of mitochondrial membrane potential

2.11. Western blotting

2.12. Statistical analysis

3. Results

3.1. BS-Z15 SMs inhibited the tumor growth in vivo

3.2. BS-Z15 SMs inhibited the growth of H22 and BEL-7404 cells in vitro

3.3. BS-Z15 SMs arrested the cell cycle of BEL-7404 cells

3.4. BS-Z15 SMs induced apoptosis in BEL-7404 Cells

3.5. BS-Z15 SMs reduced mitochondrial membrane potential (∆ψm)

4. Discussion

Declarations

References

Additional Declarations

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