Butyrate Inhibits the Mitochondrial Complex Ι to Mediate Mitochondria Dependent Apoptosis of Cervical Cancer Cells

doi:10.21203/rs.3.rs-2469528/v1

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Butyrate Inhibits the Mitochondrial Complex Ι to Mediate Mitochondria Dependent Apoptosis of Cervical Cancer Cells

https://doi.org/10.21203/rs.3.rs-2469528/v1

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Background

Cervical cancer (CC) is a common gynecological malignant tumor with high morbidity worldwide. Butyrate, a short-chain fatty acid produced by the intestinal flora, was reported to inhibit cervical carcinogenesis. This study aimed to further investigate the pro-apoptotic effects of butyrate on CC and the underlying mechanisms.

Methods

Human Hela and Caski cells were used in this study. Cell proliferation was assessed using CCK-8 and EdU staining. Cell migration and invasion were detected by Transwell and wound healing assay. The cell cycle, mitochondrial membrane potential and apoptosis were evaluated by flow cytometry. Reactive oxygen species (ROS) assay was performed for the mitochondrial function. Western blot and RT-qPCR were carried out to examine the related genes and proteins of mitochondrial complex Ι and apoptosis. The metabolite changes were analyzed by energy metabolomics and assay kits. The association between G protein-coupled receptor 41, 43, 109a and CC prognosis was analyzed using data from The Cancer Genome Atlas (TCGA).

Results

CCK-8 results showed that the butyrate treatment significantly inhibited the proliferation of CC cells, which was confirmed by EdU staining and cell cycle. The data of Transwell and wound healing assay unraveled that the migration of cervical cells was dramatically reduced after butyrate treatment. Additionally, the invasion was also observed to be decreased by butyrate. The western blot analysis showed that cleaved-Caspase 3 and cleaved-PARP, the executors of apoptosis, were increased by butyrate treatment. The results of Annexin V/PI staining and TUNEL also found the increase of apoptotic cells induced by butyrate. The expression of Cytochrome C, Caspase 9, BAX, but not Caspase 12 or 8, were up-regulated under butyrate exposure. The accumulated ROS was observed in butyrate-treated cells. Furthermore, energy metabolism showed that levels of mitochondrial NADH and NAD + decreased after butyrate treatment, which was consistent with the results of the NAD+/NADH Assay Kit. Western blot results also demonstrated that mitochondrial complex Ι reduced by butyrate.

Conclusions

Collectively, our results revealed that butyrate can inhibit the proliferation, migration and invasion of CC cells, and induce mitochondrial dependent apoptosis by inhibiting mitochondrial complex Ι.

butyrate

cervical cancer

mitochondria

apoptosis

mitochondrial complex Ι

Cervical cancer (CC) is the fourth most common cancer among women worldwide, with 570,000 new cases and 311,000 deaths every year, seriously threatening the life and health of female patients [1]. However, because of the subtle CC early symptoms, most patients are in the middle and late stages when diagnosed, with low 5-year survival rate [2]. The current treatment of CC is mainly based on surgery, supplemented by chemotherapy and other treatments including radiation, immunologic and targeting agents, which could bring permanent damage to patients [3]. The limited therapeutic effect of patients with CC is due to the incomplete knowledge of its pathogenesis. Although human papillomavirus (HPV) is a well-established cause of CC, other factors such as vaginal microbiota are likely to play an important role [4, 5]. Studies have demonstrated that the decreased Lactobacillus abundance and increased anaerobes were associated with increasing severity of CC [6, 7].

Evidences support that lactic acid bacteria plays an anti-cancer effect through various mechanisms including producing antitumor metabolites, such as lactate and short-chain fatty acids (SCFAs) [8]. Butyrate, the most biologically active SCFA, is a saturated fatty acid stemmed from the intestinal microbial fermentation of dietary fiber. It serves essential anti-inflammatory and anti-tumor roles through multiple pathways in addition to regulating intestinal flora and maintaining intestinal homeostasis [9, 10]. Butyrate was proved to activate pyruvate kinase M2 to reprogram the metabolism of colorectal cancer cells and thus suppress their proliferation in vitro experiment [11] The previous study of breast cancer showed that butyrate interfered cancer cell glycolysis to boost apoptosis attributing to the repression of breast cancer development [12] Consistently, butyrate was reported to inhibit the gastric cancer cells through enhancing the expression of tumor suppressor gene and inducing cell cycle arrest [13]. The effect of butyrate on CC has also been proposed, further studies explored the underlying mechanism and paid attention to cell apoptosis. In Hela cell line treated with butyrate for 72 h, apoptosis was verified to be significantly increased by flow cytometry [14]. Interestingly, butyrate appeared to inhibit telomerase and release more Cytochrome C (CytC) and apoptosis-inducing factor from mitochondria, which contributes to the enhanced apoptosis [15]. Increased apoptosis involved the overexpression of p53 and p73 was also observed after butyrate exposure [16]. Although butyrate was confirmed to boost apoptosis in CC cells, the exact mechanism was not clear.

Several studies have described that mitochondrial complex Ι played a significant role in cell respiration and redox [17]. And the deletion of mitochondrial complex Ι led to changes in cell homeostasis, such as ROS, mitochondrial membrane potential [18]. Further study found that deletion of mitochondrial complex Ι can induce apoptosis of human fibrosarcoma cells [19]. Mariana et al. showed that butyrate affected the energy metabolism in breast cancer cell lines and that mitochondria may occupy a central role in metastasis [20]. However, the effects of butyrate on mitochondria complex Ι and the detailed role in the butyrate-induced apoptosis of CC remain unclear.

This study aimed to evaluate the inhibitory effects of butyrate on the biological features including cell proliferation, invasion and migration of cervical cells and investigate the possible mechanisms. Our work found butyrate induce mitochondrial dependent apoptosis, and the levels of mitochondrial complex Ι was reduced in CC cells, which was the key reason. Findings from the current study will pave a solid foundation for clinical usage of butyrate to treat CC.

2.1. Reagents, Chemicals and Kits

Sodium butyrate (purity > 98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). 100 mM stock solution of butyrate was prepared, filtered with a 0.22um filter membrane and stored at -20°C. The primary antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AB-P-R001) and caspase 3 (AC0301) were from Hangzhou Goodhere Biotechnology co., LTD and Beyotime Biotechnology (Shanghai, China), respectively. The primary antibodies for cleaved-caspase 3 (9661S), caspase 9 (#9508) and BCL-2 associated X (BAX, 2772) were all purchased from Cell Signaling Technology, Inc. (Shanghai, China). And that for Caspase 8 (ET1612-70), Caspase 12 (HA500144), CytC (R1510-41), apoptotic protease activating factor 1 (Apaf-1, R1312-20) and B-cell lymphoma 2 (BCL-2, ER1706-47) were obtained from Hangzhou HuaAn Biochemical Technology Co., Ltd. (Hangzhou, China). PARP /cleaved-PARP (WL01932) was from Wanlei Biotechnology Co., Ltd (Shenyang, China). The NAD+/NADH Assay Kit (S0175) was purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China). These primary antibodies were diluted 1:1000 and stored at 4°C. The secondary antibodies were used at a concentration of 1:5000 and purchased from Proteintech Group, Inc. (Wuhan, China).

2.2. Cell Culture

Human CC cell line Hela and Caski were from the American Type Culture Collection (ATCC, Rockville, USA). Hela and Caski cell lines were cultured in Dulbecco's Modified Eagle’s Medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640, respectively, with 10% fetal bovine serum (FBS) and incubated at 37°C with 5% CO2.

2.3. CCK-8 Proliferation Assay

Cell proliferation was evaluated with a cell counting kit (CCK-8, CK04, Dojindo, Shanghai, China). 3000 cells were added in 96-well plates and cultured for 24h. Cells were then incubated with 5 mM butyrate for 12, 24, 48 and 72h. One hundred microliters of 10% CCK-8 solution were added to each microwell and the microplates were further incubated for 2h. The supernatant was harvested and the absorbance was measured at 450nm.

2.4. EdU Staining Test

Cell-Light EdU Apollo567 In Vitro Kit (C10310-1, Ribobio, Guangzhou, China) was obtained for DNA replication of cancer cells. Monolayers of Hela and Caski cells pre-grown in 12-well microplates were incubated for 24h and then treated with 5mM butyrate for 48h, followed by EdU labeling, cell immobilization, Apollo staining and DNA staining. The red fluorescence intensities were imaged using a fluorescence microscope (Leica Biosystems, Shanghai, China).

2.5. Flow Cytometry for Apoptosis and Cell Cycle Analysis

FITC Annexin V Apoptosis Detection Kit (556547, Becton Dickinson, Shanghai, China) was employed to detect the apoptosis and cell cycle of CC cells. Human CC cells were seeded into 6-well plates and treated with 5mM butyrate for 48h. Cells for apoptosis detection were collected after centrifugation and resuspended in 500µL binding buffer with 5µL of Annexin V and 5µL of propidium iodide (PI), and incubated in dark for 15 min under room temperature. But for cell cycle assay, collected cells were fixed with 75% ethanol overnight and then stained with PI. Flow cytometer (Becton Dickinson, Shanghai, China) was used for apoptosis and cell cycle analysis.

2.6. Wound Healing Assay

Hela and Caski cells were seeded into 6-well plates and grown to ~ 100%. A scratch perpendicular to the same place of each well was made by a pipette tip. 2% FBS culture medium containing 5mM butyrate or not were added, incubated at 37°C, 5% CO2 and imaged at 0, 12, 24, 48h using a light microscope.

2.7. Transwell Assay

200µL of basement membrane (Corning Life Science, Shanghai, China) diluted with phosphate buffer saline (PBS) were added in the upper chambers and incubated at 37°C, 5% CO2 for 2h to throw unnecessary PBS out. Upper chambers for migration did not need any treatment. Hela and Caski cells were collected and resuspended with serum-free medium, and then inoculated in the upper chambers. The lower chambers were filled with culture medium with 10% FBS. The chambers of Hela and Caski cells were incubated at 37°C, 5% CO2 for 8h and 17h, respectively, then fixed with 4% formalin for 30 min, 0.1% crystal violet stained for 15 min and finally observed under a light microscope.

2.8. Western Blot Analysis

CC cells were seeded in 6-well plates and collected after butyrate treatment for 48h. RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and protease inhibitor PMSF (ThermoFisher, Shanghai, China) were used for protein extraction. Concentrations of extracted proteins were determined by BCA assay kits (Beyotime Biotechnology, Shanghai, China). Equal amounts of Protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Inc. Shanghai, China). The membrane was blocked with 5% skim milk at room temperature for 1.5h and then incubated with primary antibodies overnight at 4°C on a shaker. The membranes were washed three times with Tris buffered saline with Tween (TBST), and incubated with the secondary antibody for 2h at room temperature. The band was scanned using an Amersham Image 680 imaging system and quantified by densitometry using Image J software (National Institutes of Health, Bethesda, MD, USA). The relative expressions of proteins were calculated by using GAPDH as an internal control.

2.9. TUNEL Apoptosis Analysis

TUNEL apoptosis assay kit (40307ES20, Yeasen Biotechnology Co, Ltd. Shanghai, China) was used to examine apoptosis of human CC cells. Human CC cells were seeded in 12-well plates containing climbing sheets for 24 hours. The experimental group was exposed to 5mM butyrate for 48h and then washed three times with PBS. Treated cells were fixed with 4% paraformaldehyde for 30 min, resuspended in PBS with 0.3% Triton X-100 and incubated at room temperature for 5 min. TUNEL solution (50µL) was added to the sample, followed by incubation at 37°C for 60 min in the dark. Antifading mounting medium (with DAPI, Solarbio Science & Technology Co., LTD. Beijing, China) was used for sealing and the stained CC cells were examined with a fluorescence microscope (Leica Biosystems, Shanghai, China).

2.10. ROS Production Assay

Mitochondrial reactive oxygen species (ROS) were qualitatively determined by a superoxide fluorescent probe (40778ES50, Yeasen Biotechnology Co, Ltd. Shanghai, China). Hela cells were cultured in 12-well plates containing climbing sheets for 24h. The experimental group was treated with 5mM butyrate for 48h, followed by three times of washing with PBS. ROS working solution (5uM) was added and the plates were further incubated at 37°C in the dark for 10 min. The sealed samples were examined with the Leica Biosystems fluorescence microscope.

Flow cytometry was also employed for qualitative detection of ROS. Hela cells were seeded in 6-well plates and treated with 5mM butyrate for 48h. After treatment, 5uM ROS working solution was added and incubated in dark for 10 min at 37°C. The ROS expression were detected by Becton Dickinson Flow cytometer.

2.11. CytC Expression Assay

Hela cells were seeded in 12-well plates containing climbing sheets and exposed to 5mM butyrate for 48h. After PBS wash three times, the cells were fixed with 4% paraformaldehyde for 15 min, rewashed in PBS, treated with 0.1% Triton X-100 for 5 min, blocked with 1% bovine serum albumin for 30 min and incubated with CytC primary antibodies (1:500) overnight in the dark at 4°C. Treated cells were then washed with PBS and incubated with the secondary antibody (ThermoFisher, Shanghai, China) for 2h in the dark at room temperature. Fluorescence microscope were used for the sample detection.

2.12. Real-time q-PCR

Gene expression of mitochondrial complex Ι-related was determined by real-time q-PCR (RT-qPCR). Total RNA was extracted from Hela cells, GAPDH was used as an internal control. Fold-changes were calculated by the 2^−∆∆Ct method (Primer information is shown in Supplementary table ).

2.13. Sample preparation of targeted metabolomics of energy

Hela cells were seeded in 10cm dishes and exposed to 5mM butyrate for 48h. Cells were grown to approximately 90% confluency and then harvested. After cold PBS and cold normal saline wash three times, cells were dissolved in 1ml cold 4/4/2 (v/v/v) methanol/acetonitrile/water. Then all cell samples were collected in 1.5ml centrifuge tubes stored in − 80°C refrigerator after liquid nitrogen quick freezing. Samples were separated by Agilent 1,290 Infinity UPLC system in Shanghai Applied Protein Technology Co.,Ltd. (Shanghai, China). Data were acquired using QTRAP5500 (ABSCIEX) mass spectrometry in negative ionization mode. Extraction of chromatographic peak area and retention time by Multiquanta software. Retention time corrected by energy metabolite standard.

2.14. Levels of mitochondrial NADH and NAD + Assay

NADH and NAD + concentrations were quantitatively determined by NAD+/NADH Assay Kit (Beyotime Biotechnology, Shanghai, China). Hela cells were seeded in 6-well plates and treated with butyrate or rotenone for 48h. Cells were washed two times with PBS, added to NADH and NAD + extract to lysis cell. Then centrifugated 1,2000g for 5min at 4°C to obtain cell suspension. Drew standard curve and measured the absorbance samples of at 450nm.

2.15. Flow Cytometry for Mitochondrial Membrane Potential Analysis

Mitochondrial membrane potential was detected by JC-1 fluorescent dye (Nanjing Jiancheng Bioengineering institute, Nanjing, China). Hela cells were seeded in 6-well plates and treated with butyrate for 48h. Collect cells in 1.5ml centrifuge tube and wash cells twice with PBS, prepare JC-1 working solution to suspend the cells. Then cells were incubated at 37°C, 5% CO2 for 20min, rewashed in 1X incubation Buffer, resuspended with 1×Incubation Buffer. Detection of apoptosis was by flow cytometry. Green fluorescence was detected through FITC channel, and red fluorescence was detected through PE channel.

2.16. Rotenone IC50 Assay

Detected the tolerance of Hela cells to rotenone concentration with a cell counting kit (CCK-8, CK04, Dojindo, Shanghai, China). 3000 cells were added in 96-well plates and cultured for 24h. Cells were then incubated with 0.1uM, 0.25uM, 0.5uM, 0.75uM, 1uM, 2uM rotenone for 48h. One hundred microliters of 10% CCK-8 solution were added to each microwell and the microplates were further incubated for 2h. The supernatant was harvested and the absorbance was measured at 450nm.

2.17. Statistical Analysis

Data were displayed as mean ± standard deviation (SD). Differences between two groups were evaluated using Two-sided unpaired Student’s t-test, with a P-value less than 0.05 considered statistically significant.

3.1. Butyrate Suppressed the Proliferation of Human CC Cells

Human CC cells Hela and Caski were treated with 5 mM butyrate for 12, 24, 48, 72h to observe the effects of butyrate on cell proliferation using CCK-8 detection. The results showed a significant inhibition of cell proliferation for both Hela and Caski cell lines at different time point (Fig. 1A, B). EdU staining for cellular DNA also showed a similar trend with decreased red florescence intensity of CC cell lines under butyrate exposure for 48h (Fig. 1C-F). Cell cycle distribution analysis by flow cytometry indicated that the number of cells in S-phase was obviously reduced after butyrate treatment in Hela cell line, implying that butyrate suppressed cell proliferation of CC cells by arresting the cell cycle at the G1 phase (Fig. 1G, H).

3.2. Butyrate Reduced the Invasion and Migration of Human CC Cells

Due to the invasion and migration are the important phenotype of tumors, we also evaluated the effects of butyrate on these aspects in the study. Scratch treatment was performed to observe the migration of Hela and Caski cells after treatment with 5mM butyrate. The dramatically wider distance between residual scratches in butyrate-treated CC cells was found at 24h and 48h time point (Fig. 2A-D). Furthermore, the Transwell experiment was employed and the results revealed that butyrate remarkably decreased cell invasion and migration in both Hela and Caski cells (Fig. 2E-G).

3.3. Butyrate Promoted Apoptosis of Human CC Cells

The results of TUNEL staining demonstrated that green fluorescence intensity of butyrate-treated Hela was significantly increased, indicating that butyrate exactly enhanced CC cell apoptosis (Fig. 3A). Annexin V /PI staining and flow cytometry were further employed to quantitate apoptosis of Hela cell line. The early (Annexin V +, PI -) and late (Annexin V +, PI +) apoptosis rate of Hela cell were 22.6% and 22.7% in butyrate group and 12.1% and 6.1% in the control group (Fig. 3B). We further investigated the molecular changes that underlie the inhibitory effect of butyrate on CC cells. As the cleaved-Caspase 3 and cleaved-PARP are the key executive factors for apoptosis, it was detected using western blot analysis and observed with a significant increase after butyrate treatment (Fig. 3C-F).

3.4. Butyrate Activated Mitochondria Dependent Apoptosis Pathway

The western blot results demonstrated that butyrate up-regulated the expression of Caspase 9, but not Caspase 8 or 12 (Fig. 4A, B), indicating that the mitochondria-dependent pathway is the response for the inhibition effects of butyrate on human CC. Moreover, the notable elevation of Apaf-1 and CytC, the key parts of mitochondrial apoptosis complex, were found after 48h of butyrate exposure using western blot and fluorescence staining (Fig. 4C-E). In addition, the synthesis of pro-apoptotic protein BAX increased while anti-apoptotic protein BCL-2 remained unaltered after butyrate treatment (Fig. 4D, E). ROS assessment using fluorescence staining and flow cytometry showed a higher production of intracellular ROS in CC cells receiving butyrate treatment (Fig. 4F, G).

3.5 GPR may not be the key mechanism of butyrate anti-CC effect

TCGA data showed no difference in the expression of butyrate receptors GPR41(Fig. 5A), 43 (Fig. 5B) between human CC tumor tissue and normal tissues, but not significant difference in GPR109a (Fig. 5C). For GPR43 109a, there was no difference in the expression when CC stage developed. We further re-analyzed TCGA data for correlation between butyrate receptors and the prognosis of GC patients. The prognosis of CC patients was not changed when patients expressed high or low at GPR41, 43, 109a. These results suggested that butyrate receptors GPR41, 43, 109a might not be a key contributor of the anti-tumor effects of butyrate on CC.

3.6 Butyrate reduced the levels of mitochondrial NADH and NAD + in CC

Study showed that mitochondrial-dependent apoptosis was closely related to mitochondrial function. We detected mitochondrial energy metabolite in Hela cells (Fig. 6A), and found that levels of mitochondrial NADH and NAD + were decreased significantly after treatment with 5mM butyrate (Fig. 6C). In addition, the level of NDUFA1, NDUFB1, NDUFC1 mRNA compensatory increased while NQO1 was decreased, causing oxidative stress damage (Fig. 6B). Mitochondrial membrane potential, a key factor for maintaining mitochondrial function, was decreased in Hela cells after butyrate exposure (Fig. 6D).

3.7 Inhibition of mitochondrial complex Ι induced mitochondrial-dependent apoptosis in CC

Treatment of Hela cells with rotenone to determine optimum concentration (Fig. 7A). We found that butyrate and rotenone could reduce NADH, NAD + production and mitochondrial complex Ι expression (Fig. 7B, E). Moreover cleaved-Caspase 3, was detected using western blot analysis and observed with a significant increase after rotenone treatment (Fig. 7D, F). The key proteins of mitochondrial apoptosis Caspase 9, BCL-2, Bax, Apaf-1, were significantly increased. Immunofluorescence also showed a higher production of intracellular ROS receiving rotenone treatment (Fig. 7C).

Butyrate, an essential mediator in gut microecology, has been authenticated to inhibit a variety of malignant tumors [21]. The current study on CC found that butyrate dramatically suppressed the proliferation of CC cells, reduced cell migration and invasion via enhancing apoptosis. The TCGA data showed butyrate receptors may not be a key factor. Furthermore, the data indicated that butyrate-induced apoptosis was mediated by endogenous mitochondria rather than exogenous death receptor or endoplasmic reticulum stress, and the reduction of mitochondrial complex Ι was the main cause of butyrate-induced mitochondria apoptosis.

Butyrate has been identified as an anti-tumor chemical, capable of inhibiting the proliferation of tumor cells such as lung cancer cells, esophageal cancer cells, liver cancer cells, gastric cancer cells and CC cells in vitro [22–25]. Consistently, our results also confirmed that the growth of Hela cells was inhibited and arrested in G1 phase. A previous study showed that butyrate induces G1 phase arrest of Hela cells by up-regulating the expression of p21 and down-regulating cyclin D1 [26]. Due to the migration and invasion of CC are recognized to contribute to poor prognosis and therapeutic outcomes of patients [27], we further employed the Transwell experiment and found that butyrate treatment significantly decreased the migration as well as invasion in Hela cell line and Caski cell line. Since triggering apoptosis has become an important mechanism for butyrate exerting its anti-cancer effects [28], we further detected the changes of apoptosis-related parameters after butyrate treatment. In line with the result of Annexin V/PI staining, the TUNEL staining also showed that butyrate exposure increased the apoptotic cells. The western blot analysis consistently proved that cleaved-Caspase 3, cleaved-PARP were increased by butyrate treatment. The previous researches showed that butyrate could increase the synthesis of pro-apoptotic protein BAX but decreased the anti-apoptotic BCL-2 to promote mitochondria dependent apoptosis [29, 30]. However, our data showed butyrate only regulated the expression of BAX protein not BCL-2 in CC cells. The accumulation of ROS is considered as the early event of mitochondria mediated apoptosis [31], which was confirmed by our findings. We also found Apaf-1 and CytC, the key factors mediating mitochondrial pathway of apoptosis [32], were significantly up-regulated after butyrate treatment, which was in line with the study of Tiwari et al [33]. Our western blot results showed that Caspase 9 was significantly increased, but not Caspase 8 or Caspase 12. Collectively, our data indicated that butyrate treatment activated the mitochondria dependent apoptosis of human CC.

NADH and NAD + as essential energy metabolism coenzymes in cancer, were the key factor of cancer development [34]. Studies have shown that the NAD+/NADH balance can inhibit the aggressiveness of human breast cancer cells [35], NAMPT-mediated upregulation of the NADH and NAD + protects colon cancer cells against detrimental oxidative stress [36]. However, it remains unclear whether NADH and NAD + could be used as therapeutic target for human CC. Our metabolic group showed that the levels of NAD + and NADH in Hela cells were significantly decreased after butyrate treatment. Importantly, mitochondrial complex I dysfunctions was attributed to decreased coenzyme (NADH and NAD+) activity [37]. When we used mitochondrial complex Ι inhibitor, rotenone, to treat Hela cells, it activated mitochondrial apoptosis pathway and promoted ROS accumulation. Overall, our results showed that the inhibition of mitochondrial complex Ι was the main mechanism for butyrate induced apoptosis.

“Butyrate paradox” describes the opposite effects of butyrate on cancer cells and normal colonic epithelium. As the necessary nutrition, butyrate is optimally utilized to repair damaged epithelial cells to maintain the intact of lumen mucus but inhibit the cancerous cells [38]. Previous scholars showed that small amount of butyrate was metabolized to acetyl-CoA to stimulate histone acetyl transferases (HATs) while high level accumulated in nucleus and acts as a histone deacetylase inhibitor (HDACi) [39]. Both mechanisms increased histone acetylation to regulate the expression of different genes, thereby causing a wide range of anti-tumor effect including inducing cell apoptosis and reducing proliferation [40]. Further studies revealed that butyrate could reduce the expression of anti-apoptotic genes such as BCL-2, BCL-xl, while increasing the pro-apoptotic genes including BAX, BAK, thereby enhancing the cell apoptosis [29, 41]. Butyrate, on the other hand, altered the expression of genes involved in metabolism, which was the fundamental mechanism behind Warburg effect, in which cancer cells employed glycolysis rather than oxidative phosphorylation for energy requirements under oxygen condition. Butyrate was proved to significantly inhibit the glucose metabolism by decreasing the mRNA levels of glucose transporter GLUT1 and glucose-6-phosphate dehydrogenase, thereby inducing more apoptotic colorectal cancer cells [42]. In addition, butyrate could interact with G protein-coupled receptor 41 (GPR41), GPR43 and GPR109a to play anti-inflammation and anti-tumor effects. GPR109a was shown to bind with its ligand butyrate and induce cell apoptosis in colon cancer cells [43]. In breast cancer, butyrate could activate GPR109a to suppress the expression of cell survival and anti-apoptotic genes [44]. However, we found that the expression of G protein coupled receptors in human CC is not obvious, and its detailed mechanism remains to be further studied in the future.

Interestingly, Hela and Caski cell lines used in our study are HPV-18 and HPV-16 positive, respectively, suggesting the potential therapy of butyrate on CC with HPV infection. Although HPV is known as the important etiological factor of CC, cervical epithelium carcinogenesis was dependent on vaginal microbiota in the HPV positive patients [45]. Studies have revealed unique microbial signatures associated with cervical carcinogenesis. For HPV-positive woman, the bacterial diversity and composition were more complex compared to HPV-negative woman [46–48]. Moreover, investigations have showed that cervical microbiota like Fusobacterium may increase anti-inflammatory cytokines to play an important role in the development of immunosuppressive microenvironment related to HPV infection and persistence [49]. Chlamydia trachomatis was proved to increase HPV infection to persist, and the coinfection seemed to cause CC at higher risk [50, 51]. Butyrate was found to correct the dysbiosis of intestinal microbiota, and increased the abundance of the beneficial bacteria including Lactobacillus [52]. As known, butyrate is the important metabolite of Lactobacillus and Fusobacterium, indicating that butyrate might play a critical role for vaginal microbiota against CC. Notably, early study in 1998 has demonstrated the direct suppression effect of butyrate on the replication of HPV-11 [53].

In conclusion, our results revealed that butyrate suppressed the cell proliferation, invasion and migration of CC cell lines through reducing mitochondrial complex I to activate mitochondria dependent pathway of apoptosis. Given the inhibition effect of on HPV positive CC cells, butyrate may be with the potential therapeutic effect on HIV-positive CC patients.

Availability of data and materials

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Funding

This study was supported by the Science and Technology Department of Zhejiang (No. LGF20H070003), the Medical and Health Research Project Grant of Zhejiang Province of China (No. Y2019317606), the Technology Bureau of Wenzhou (No. Y2020214 and Y20190060) and P&PMed (Shanghai) Biotechnology Co., Ltd.

Authors' contributions

Conceptualization—Guo Junping and Xiangyang Xue; Methodology—Ke Zhang and Xiawei Ji; Formal analysis—Zhengyang Song and Tiantian Yu; Investigation—Dibang Shi; Writing and original draft preparation—Ke Zhang, Xiawei Ji and Fangyan Wang; Writing, review, and editing—Fangyan Wang and Guo Junping. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

The authors would like to extend thanks to all those who participated in this research.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that there are no conflicts of interest.

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No competing interests reported.

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Butyrate Inhibits the Mitochondrial Complex Ι to Mediate Mitochondria Dependent Apoptosis of Cervical Cancer Cells

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Abstract

Background

Methods

Results

Conclusions

Figures

1. Introduction

2. Materials And Methods

2.1. Reagents, Chemicals and Kits

2.2. Cell Culture

2.3. CCK-8 Proliferation Assay

2.4. EdU Staining Test

2.5. Flow Cytometry for Apoptosis and Cell Cycle Analysis

2.6. Wound Healing Assay

2.7. Transwell Assay

2.8. Western Blot Analysis

2.9. TUNEL Apoptosis Analysis

2.10. ROS Production Assay

3. Results

3.1. Butyrate Suppressed the Proliferation of Human CC Cells

3.2. Butyrate Reduced the Invasion and Migration of Human CC Cells

3.3. Butyrate Promoted Apoptosis of Human CC Cells

3.4. Butyrate Activated Mitochondria Dependent Apoptosis Pathway

3.5 GPR may not be the key mechanism of butyrate anti-CC effect

3.6 Butyrate reduced the levels of mitochondrial NADH and NAD + in CC

3.7 Inhibition of mitochondrial complex Ι induced mitochondrial-dependent apoptosis in CC

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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