Chitopentaose Inhibits Hepatocellular Carcinoma by Inducing Mitochondrial Mediated Apoptosis and Suppressing Protective Autophagy

doi:10.21203/rs.3.rs-94944/v1

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Chitopentaose Inhibits Hepatocellular Carcinoma by Inducing Mitochondrial Mediated Apoptosis and Suppressing Protective Autophagy

https://doi.org/10.21203/rs.3.rs-94944/v1

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published 05 Jan, 2021

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Hepatocellular carcinoma (HCC) is one of the most prevalent and deadliest cancers. In this study, the anti-tumor effect of singular degree of polymerization (DP) chitooligosaccharides (COS) (DP 2-5) and the underlay molecular mechanisms were investigated on HCC cell line HepG2. MTT assay showed that (GlcN)₅ have the best anti-proliferation effect among the different DP of COS (DP2-5). Furthermore, the administration of (GlcN)₅ could decrease mitochondrial membrane potential, release cytochrome c into cytoplasm, activate the cleavage of Caspases9/3, thus inducing mitochondrial-mediated apoptosis. Additionally, (GlcN)₅ treatment could increase the accumulation of autophagosomes. Further investigation showed that (GlcN)₅ suppressed protective autophagy at the fusion of autophagosomes and lysosomes. Moreover, the inhibition of protective autophagy flux by (GlcN)₅ could further decrease cell viability and increase apoptosis rate. Our findings suggested that (GlcN)₅ suppressed HepG2 proliferation through inducing apoptosis via intrinsic pathway and impairing cell protective autophagy. COS might have the potential to be an agent for lowering the risk of HCC.

Biotechnology and Bioengineering

chitooligosaccharides

singular DP

HCC

apoptosis

autophagy

Hepatocellular carcinoma (HCC) ranks fifth in the male cancer patients and the ninth in female cancer patients around the world, as well as high mortality rate among all cancer cases(Stewart & Wild, 2014). The main causes of HCC are the chronic diseases due to hepatitis B virus infection in south-east Asia and sub-Saharan Africa, hepatitis C virus infection in the United States, and the abuse of alcohol(El-Serag, 2011). Current treatment against HCC include liver transplantation, surgical resection, local ablation, and targeted molecular therapy such as Sorafinib, a multikinase inhibitor against vascular epithelial growth factor receptor-2/3 and raf-kinase(Rahbari et al., 2011). However, traditional therapies are restricted by liver source and tumor reoccurrence, and patients receiving targeted molecular therapy suffered from several side effects such as fatigue, diarrhea and hand-foot skin reaction(Dong & Roberts, 2010). Additionally, tumor cells are easily to resistant against drugs(Hwang et al., 2007). Therefore, the prevention of HCC largely depends on the successful treatment with safe, efficient, high-quality, and searching for alternative effective agents is urgently needed.

Recently, phytochemical combined cancer therapies have received a great deal of attention. Chitooligosaccharides (COS) are the hydrolysis product of chitosan, which is the deacetylation derivate of chitin, a polysaccharide found in the exoskeleton of shrimps and crabs, and the cell wall of fungi. It is suggested that COS exert multiple bioactivities including anti-inflammatory(Bahar, O’doherty, Maher, McMorrow, & Sweeney, 2012), anti-microbials(Jumaa, Furkert, & Muller, 2002), anti-Alzheimer’s disease(Jia et al., 2016), anti-obesity(X. Li et al., 2018), anti-oxidative stress(Qiao, Bai, & Du, 2011), wound healing(Ueno et al., 1999), and improving plant defense response(He et al., 2018). Since 1986, COS were reported to inhibit tumor in vivo(Suzuki et al., 1986), and the anti-cancer property of COS has received high attention. Previous studies had revealed that COS could inhibit the progression of several cancers including colon carcinoma, cervical cancer and gastric cancer (Z. Luo, X. Dong, Q. Ke, Q. Duan, & L. Shen, 2014; Zhao et al., 2019; Zou, Yuan, Yang, Zhai, & Wang, 2018). However, most of these studies were performed with the combination of COS with different degree of polymerization (DP), and the anti-tumor effect of COS with singular DP was quite unclear.

Inducing apoptosis is a common strategy for inhibiting cancer progression(Wong, 2011). Moreover, it was reported that the inhibition of autophagy could enhance apoptosis in HCC(Longo et al., 2008). Indeed, the crosstalk of autophagy and apoptosis impacts the progression of tumor and therefore is highly concerned(Eisenberg-Lerner, Bialik, Simon, & Kimchi, 2009). However, it remains unknown whether the crosstalk of apoptosis and autophagy is involved in the COS mediated anti-cancer effect, as well as how it works.

Here, we hypothesized that COS with certain DP could induce mitochondrial mediated-apoptosis and impair cell protective autophagy. This research was designed to determine the effect of singular DP COS on HCC cell proliferation invitro and explore the role of COS in the regulation of apoptosis and autophagy.

Cell lines and reagents

Human liver epithelial HepG2 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco (Gaithersburg, MD). Dulbecco’s Modified Eagle Medium (DMEM) was purchased form HyClone (Logan, UT). Antibodies against cleaved cysteinyl aspartate specific proteinase (caspase)-9, cleaved caspase-3, Bcl2, Bax, β-actin, and microtubule-associated proteins 1A/1B light chain 3B (LC3B) were obtained from Cell Signaling Technology (Danvers, MA). Anti-poly ADP-ribose polymerase-1 (anti-PARP-1) was provided form Abcam (Cambridge, UK). Anti- SQSTM1/p62, anti-Beclin1, and anti-lysosomal-associated membrane protein 1 (anti-LAMP1) were purchased from Proteintech (Rosemont, IL). 3-methyladenine (3-MA) and chloroquine (CQ) were purchased from MedChem Express (Princeton, NJ). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and other chemical agents were of the analytical grade.

Characterization of COS

The separation of COS and high performance liquid chromatography (HPLC) were described previously in our recent work(X. Li et al., 2018). The molecular weight of COS was detected using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS, Biosystems 4700 Proteomics Analyzer, Applied Biosystem Inc., Foster City, CA). 2,5-dihydroxybenzonic acid was used as matrix. ¹H nuclear magnetic resonance (NMR) was performed on Bruker Avance-600 NMR spectrometer (Billerica, MA). COS was dissolved in D₂O. Fourier transform–infrared (FT-IR) spectrometer (Nicolet 6700, Nicolet Instrument Co., Madison, WI) was used to detect the structure of chitopentaose (GlcN)₅.

Cell culture and viability assay

HepG2 cells were cultured in DMEM supplemented with 10% FBS in humidified atmosphere with 5% CO₂ at 37^oC. The viability of HepG2 cells were measured by MTT assay as described previously(Mosmann, 1983). Briefly, cells were seeded into a 96-well plate at a density of per well for 24 h, and then exposed to different concentration (1-7 mg/mL) of COS with different DP. Afterwards, a solution containing 100 μg MTT were added to each well and incubated for 4 h at 37^oC. After removing the solution, the formazan was dissolved with 150 μL of DMSO. The absorbance at 570 nm was read on a microplate reader (Tecan, Zürich, Switzerland). The cell viability was defined as the percentage of the control group. The calculated 50% inhibition concentration (IC₅₀) value was calculated using Graphpad Prism (Ver. 7.0, Graphpad Software, La Jolla, CA) through non-linear regression.

Determination of HepG2 apoptosis

Annexin V-fluorescein isothiocyanate isomer I (FITC)/propidium iodide (PI) dual staining

Annexin V-FITC/PI staining was conducted to detect the apoptotic cells. Briefly, cells were seeded in a 6-well plate at a density of per well over night and then exposed to COS. After 48 h, cells were harvested and centrifuged at 1000 g for 5 min and washed with phosphate buffer saline (PBS) once. Afterwards cells were suspended with 200 μL of staining buffer. 5 μL of Annexin V-FITC and 5 μL of PI were added and cells were incubated in the dark room for 20 min. Finally, 300 μL of staining buffer was added. The apoptotic cells were determined using a flow cytometry (BD Bioscience, Franklin Lakes, NJ).

DAPI staining and morphological analysis

Cells were seeded in a 24-well plate at a density of per well and treated with COS for 48 h. Culture medium was removed and cells were fixed with 4 % (v/v) paraformaldehyde for 20 min at room temperature. After treatment, cells were stained with DAPI solution (Beyotime, Shanghai, China) for 20 min at room temperature. The nuclear morphological analysis was determined using an inverted fluorescence microscope (Nikon, Tokyo, Japan).

Detection of mitochondrial membrane potential

The mitochondrial membrane potential was measured using JC-1 mitochondrial membrane potential (MMP) assay kit (Yeasen, Shanghai, China). As brief, HepG2 cells were seeded in a 96-well plate and treated with COS for different dosages for 48 h. JC-1 staining solution was added to each well and the cells were incubated at 37 ^oC for 20 min. Afterwards, the supernatant was removed, and each well was washed twice. The red (excitation 490nm, emission 530nm) and green (excitation 525nm, emission 590nm) fluorescence was detected using a microplate reader. The MMP was represented as the ratio of green/red fluorescence.

Determination of cell autophagy

Transmission election microscopy (TEM)

Cells were planted in a 6-well-plate. After being incubated with or without CQ for 2 h, cells were treated with COS for 48 h. After being harvested, cells were fixed with 4% glutaraldehyde overnight at 4 ^oC and washed 3 times with PBS. Afterwards, cells were fixed with 2% osmium tetroxide for 4 h at 4 ^oC and dehydrated with gradient ethanol. After being embedded and selection, the cells were stained with uranyl acetate and led acetate. The samples were observed using an electron microscope (JEOL, Tokyo, Japan).

Transfection of mRPF-GFP-LC3

The transfection of mRFP-GFP-LC3 and detection of the fusion of LC3 and lysosomes were performed using a mRFP-GFP-LC3 adenovirus vector kit (Hanbio, Shanghai, China) according to the manufacture’s instruction. Briefly, HepG2 cells were seeded in a 24-well plate and incubated with adenovirus to transfect mRFP-GFP-LC3 vector for 6 h. After being treated with 3-MA or CQ for 2 h to block the autophagy flux, the cells were incubated with chitopentaose for 48 h. The fluorescence signals were detected using an inverted fluorescence microscopy.

Colocalization of LC3 and lysosomes

Immunofluorescence was used to detect the colocalization of LC3 and lysosomes. Cells were seeded in a 24-well plate and pre-treated with or without CQ for 2 h and then treated with COS for 48 h. After removing the medium, cells were fixed with methanol for 20 min on ice and rinsed with PBS for 3 times. The cells were blocked with PBS containing 5% FBS and 1% Triton 100 and incubated with primary antibody at 4 ^oC overnight. After being washed 3 times with PBS, the cells were incubated with FITC-labeled or Alexa Fluor 594-labeled secondary antibody (Yeasen, Shanghai, China) for 1 h at room temperature. Anti-fade mounting medium (Beyotime, Shanghai, China) was used to preserve the fluorescence signals. The fluorescence signals were detected using an inverted fluorescence microscope. The colocalization analysis was conducted through ImageJ software (ver. 1.52a, Wayne Rasband, NIH). Pearson correlation coefficient was used to represent the colocalization of red and green fluorescence signals.

RNA extraction and quantitative polymerase chain reaction (qPCR) assays

Cells were seeded in a 6-well plate at a density of per well over night and then exposed to chitopentaose for 48 h. RNA was extracted using RNA extraction kit (Promega, Fitchburg, WI) according to the manufacturer’s guidance. Primers were synthesized through Sagon (Shanghai, China). Reverse transcription was conducted using a reverse transcription kit (Promega, Fitchburg, WI) according to the manufacture’s instruction. qPCR was conducted on a real-time thermal cycler (Bio-rad, Hercules, CA). The relative amount of target mRNA was calculated by the comparative cycle threshold (C_t) method (2^−ΔΔCt) by normalizing target mRNA C_t values to those for β-actin.

Protein extraction and western-blot.

For total protein extraction, after being treated with different concentration of chitopentaose, HepG2 cells were lysed with a RIPA (Thermo, Waltham, MA) and 1 mM PMSF (Beyotime, Shanghai, China) cocktail using an ultrasonic cell disruptor. Separation of cytosolic protein and mitochondrial protein was performed using a cytosol-mitochondrial isolation kit (Beyotime, Shanghai, China). Protein concentration was analyzed using a BCA kit (Beyotime, China). Equal amount of protein was electrophoresed by 12.5-15% sodium laurylsulfonate (SDS) gel and transferred to polyvinylidene fluoride membrane by the wet transfer method (Bio-rad, Hercules, CA). The membrane was blocked with TBST (20 mM Tris-base, 150 mM NaCl, 0.1% Tween-20) containing 5% (m/v) non-fat milk powder and incubated with primary antibody at proper dilutions overnight at 4 ^oC. After being washed 3 times with TBST, the membrane was incubated with secondary antibody for 1 h at room temperature. The immunoblot on the membrane was developed with enhanced chemiluminescence system. β-actin was used as an internal reference. The relative protein concentration was expressed as folds of the control group. The intensity of the immunoblot was quantified with ImageJ software.

Statistical analysis

All the experiments were replicated for more than three times. The data were expressed as mean standard deviation (SD). Statistical analysis was performed on SPSS (Ver. 22 SPSS Inc., Chicago, IL). One-way/two-way analysis of variance (ANOVA) and t-test were performed to evaluate the data. Significances were considered to exist at level of p 0.05.

Characterization of (GlcN)₅

The result of MALDI-TOF-MS analysis was shown in Figure 1a. The mass/charge (m/z) of (GlcN)₅ was 864.3 mass units corresponding to its sodium ionized-peak [M+Na⁺]. The results of ¹H NMR analysis was shown in Figure 1(b). There were absorbance peaks at 5.30 ppm (H-1α), 4.74 ppm (H-1), four absorbance peaks at 3.91-3.34 (H-3, H-4, H-5, H-6), absorbance peaks at 2.89-3.92 ppm (H-2), which was consistent with previous literature(Sugiyama et al., 2001). The HPLC result (Figure 1c) showed one peak and the retention time was 26.2 min. According to our more recent studies (Chen et al., 2019), the FT-IR results showed peaks at 1000-1200 cm^-1 (C-O-C stretching), peaks at 1622.19, 1517.23, and ~1390.62 cm^-1 were amide I, amide II and amide III, respectively. Peaks at 2910.91 and 3412.52 cm^-1 were C-H stretching and O-H stretching. These results indicated that the purity of (GlcN)₅ was above 90% with high deacetylation.

Anti-proliferation effect of COS

HepG2 cells were incubated with different concentrations of COS (DP 2-5) for 48 h. As shown in Figure 2, the anti-proliferation effect of COS was showed in a dose- and DP-dependent manner. The IC₅₀ values of different singular DP COS suppressing HepG2 cells were 7.18 0.11, 7.52 0.26, 6.49 1.05, and 3.49 0.40 mg/mL, respectively. (GlcN)₅ showed a better inhibition effect against HepG2 cells in comparison with other singular DP COS. These data suggested that COS could suppress HepG2 proliferation, but each singular DP COS had different inhibitory effect on HepG2. Various previous researches showed the anti-proliferation effect of COS on tumor cells. The relative cell viability when treated with 1 mg/mL COS for 48 h were reported ranging from 65% to 90%(Z. G. Luo, X. X. Dong, Q. Ke, Q. W. Duan, & L. Shen, 2014; Shen, Chen, Chan, Jeng, & Wang, 2009; Xu et al., 2008). All these reported generally used a complicated COS mixture (DP 2-9, for example(Xu et al., 2008)), which had different characteristic including degree of N-acetylation (DA), DP, and the mean molecular weight. The characteristic of COS is highly responsible for their bioactivities. Our results showed that DP was an important factor in the inhibition effect of COS against tumor cells, and (GlcN)₅ showed better inhibitory effect on HepG2 cells compared with (GlcN)_1-4.

COS induced apoptosis to HepG2 cells

The unbalance between cellular apoptosis and proliferation regulates the cancer pathological progression(Fong et al., 2006). Therefore, inducing apoptosis might be a strategy to inhibit tumor progression. In order to investigate whether COS would induce apoptosis, Annexin V-FITC/PI staining was conducted. The cross gate separated the cells into four groups: normal cells (FITC^-/PI^-), early apoptosis cells (FITC⁺/PI^-), late apoptosis cells (FITC⁺/PI⁺), and mechanically damaged cells (FITC^-/PI⁺). As shown in Figure 3a, after being incubated with COS, the proportion of apoptosis cells (early apoptosis + late apoptosis) was increased with the increase of DP, and (GlcN)₅ induced a significant increase in apoptotic cells (p 0.05), accounting for 24.57 2.25%. Thus, the possible mechanism of the suppression activity was investigated by using (GlcN)₅in the following research.

Chromatin condensation and DNA fragmentation are the hallmarks of apoptosis(Liu et al., 1998). As shown in Figure 3b, the white arrows pointed out the cells underwent chromatin condensation or DNA fragmentation. Compared with control group, (GlcN)₅ induced morphological changes in cell nuclear in a dose-dependent manner. These findings suggested that COS could induce apoptotic phenotype in HepG2 cells. Several natural compounds were reported to increase apoptotic phenotype in tumor cells including nuclear condensation and therefore having the potential intervention effect against cancer(H. Li et al., 2018; Salma et al., 2009).

COS activate mitochondrial-caspase apoptosis

Bcl-2 family proteins are highly involved in the progression of apoptosis (Adams & Cory, 1998). The ratio of anti and pro-apoptosis protein in Bcl-2 family is a key indicator to evaluate the effect of apoptosis stimuli. Real-time quantitative PCR was performed to detect the relative expression of bax and bcl-2. After being treated with (GlcN)₅, the relative expression of the pro-apoptotic genes, bax, increased significantly (p 0.05), while the anti-apoptotic genes, bcl-2, showed no significant change. The ratio of bax/bcl-2 increased significantly (p 0.05) in a dose-dependent manner, indicating that cells underwent apoptosis (Figure 4a). In line with the qPCR results, the relative expression of Bcl-2 decreased significantly, while Bax increased significantly after being treated with (GlcN)₅. Thus, the ratio of Bax/Bcl-2 increased significantly (p 0.05) by using western blot confirmation (Figure 4d), suggesting that cells treated with (GlcN)₅ showed an apoptotic tendency.

A decrease in MMP would result in the structural change in mitochondria, and consequently leads to the redistribution of cytochrome c(Gottlieb, Armour, Harris, & Thompson, 2003). JC-1 staining kit was used to examine the change of MMP. As shown in Figure 4b, the ratio of green/red fluorescence decreased significantly with a dose-dependent manner, suggesting MMP was decreased after (GlcN)₅treatment. The release of cytochrome c from mitochondria to cytoplasm was also detected by western blot. As shown in Figure 4c, the relative expression of cytoplasmic cytochrome c of (GlcN)₅ groups were significantly increased compared with control group (p<0.05). In line with a previous study, a polypeptide extracted from Ciona savignyi had similar effect with (GlcN)₅that depolarized mitochondria, released cytochrome c into cytoplasm, and induced apoptosis(Cheng et al., 2012).

The release of cytochrome c from mitochondria to cytoplasm could activate pro-caspase-9, which consequently cleave other caspase family proteins and finally executes cell to apoptosis(Green & Reed, 1998; Hengartner, 2000). As Figure 4d shown, the relative expression of cleaved caspase-3, cleaved caspase-9, and cleaved PARP increased significantly (p<0.05) when cells were treated with (GlcN)₅. These findings above revealed that (GlcN)₅ could alter the expression of Bcl-2 family protein, decrease the mitochondrial membrane potential, release cytochrome c from mitochondria to cytoplasm, led the cleavage of caspase-3 and caspase-9, which cleavage PARP, and finally induced apoptosis in HepG2 cells. Xu et al reported similar results, that COS could increase the cleavage of PARP(Xu et al., 2008), however, whether the proteins in caspase family were involved in the cleavage of PARP was not illustrated in their study. In this paper, we demonstrated that (GlcN)₅ induced PARP cleavage in HepG2 cells via mitochondria-caspase pathway.

COS induced autophagy markers

Autophagy plays a dual role during physiological process and tumorigenesis including maintenance of cell survival and defeat of cell malfunction (Hippert, O'Toole, & Thorburn, 2006; Mizushima, 2007). It was reported that the inducing(Yo, Shieh, & Hsu, 2009) and suppression(Law et al., 2014) of autophagy could both inhibit tumor cell viability. In the present study, we further investigated the effect of (GlcN)₅ on autophagy in HepG2 cells. TEM is a golden standard to detect the formation of autophagosomes. As shown in Figure 5a, after being treated with (GlcN)₅, HepG2 showed an increase in the formation of autophagosomes compared to control group. Meanwhile, the markers of autophagy include the conversion of LC3-I to LC3-II which indicates the accumulation of autophagosomes. In the present study, after cells being treated with (GlcN)₅, the green dots increased compared with control group, suggesting that (GlcN)₅ could accumulate autophagosomes (Figure 5b). These data were consistent with the blots of LC3B (Figure 5c), in which LC3-II expression increased in a dose-dependent manner after (GlcN)₅treatment. Additionally, the Beclin1 showed no significant change, and the p62 blots increased significantly (p 0.05) compared with control group, which suggested that the (GlcN)₅ treatment might have no effect on the formation of autophagosomes but suppress the degradation of autophagosomes.

COS suppressed autophagy flux

The accumulation of autophagosomes might be caused by the increase of formation of autophagosomes or by the reduction of the degradation of autophagosomes(Meng et al., 2018). To further investigate these two possibilities, two chemical inhibitors were co-incubated with (GlcN)₅: CQ, an inhibitor of the fusion of autophagosomes and lysosomes, and 3-MA, an inhibitor of the formation of autophagosomes.

The TEM results showed that in comparison with control group, the formation of autophagosomes increased in HepG2 cells treated with CQ alone group and (GlcN)₅ group (Figure 6a). Moreover, the co-incubation of CQ and (GlcN)₅ group induced an increase of the numbers of autophagosomes, suggesting (GlcN)₅ might have similar effect with CQ on autophagy, which suppressed the degradation of autophagosomes. This was further confirmed by the western blot assay. As shown in Figure 6b, the p62 and LC3-II blot increased as the administration of CQ, almost the same as (GlcN)₅ group, when compared to control group; while the increase was even more significant (p<0.05) when the cells were co-incubated with CQ and (GlcN)₅.

Furthermore, we checked the autophagic flux after CQ and/or (GlcN)₅ treatment by using mRFP-GFP-LC3 vector system in HepG2 cells. In this study, the LC3 proteins were labeled with red and green fluorescence protein, respectively. When the fusion of lysosomes and autophagosomes occurs, the green fluorescence quenches due to the decrease of pH in lysosomes and only the red dots remain. Conversely, yellow dots (the merge of green dots and red dots) emerged when the autophagy flux was suppressed at the late stage. As Figure 6c shown, the treatment of (GlcN)₅ led to an increase of yellow dots, in consistent with the CQ group, indicating the suppress of autophagy flux at the late stage. The co-incubation of CQ and (GlcN)₅ resulted a significant (p<0.05) increase in the yellow dots when compared with control group, CQ group and (GlcN)₅ group, showing a synergistic effect. Additionally, 3-MA could not inhibit the accumulation of yellow dots when co-treated with (GlcN)₅, suggesting that (GlcN)₅ might not increase the formation of autophagosomes. To further explore whether (GlcN)₅ interfere the fusion of autophagosomes and lysosomes, colocalization analysis of LC3 puncta and lysosomes was conducted by immunofluorescence. LC3B proteins were labeled with green fluorescence, representing autophagosomes, and LAMP1 proteins were labeled with red fluorescence, representing lysosomes. As shown in Figure 6d, with the treatment of (GlcN)₅, the Pearson correlation coefficient of LC3B and LAMP1 decreased significantly (p<0.05), indicating low colocalization. Similar situations were found in CQ group and co-administration group (CQ + (GlcN)₅). These results suggest (GlcN)₅ inhibited the fusion of lysosomes and autophagosomes and impaired autophagy flux at the late stage, finally suppressed the autophagy in HepG2 cells.

The role of autophagy in cell viability and apoptosis

The role of autophagy on tumor progression varies according to cell type and cancer stage (Glick, Barth, & Macleod, 2010). On one hand, autophagy prevents tumorigenesis through reducing inflammatory and necrosis. On the other hand, it also protects tumor through promoting cell viability under stress. In this study, we further evaluated the crosstalk between autophagy and apoptosis in HepG2, as well as investigated the role of autophagy in cell viability and apoptosis. As Figure 7a showed, cell viability decreased significantly (p<0.01) with the CQ treatment. The co-incubation of CQ and (GlcN)₅ suppressed the proliferation of HepG2 cells significantly (p<0.01) when compared with CQ group and (GlcN)₅group, suggesting the role of autophagy was to protect HepG2 cells from death. In accordance with the annexin V-FITC/PI dual staining assay (Figure 7b), the co-incubation of CQ and (GlcN)₅ induced significant apoptotic phenotype, suggesting the inhibition of autophagy induced apoptosis. In western blot assay for further substantiation (Figure 7c), the co-incubation of CQ and (GlcN)₅ induced a significant increase (p<0.05) in the relative expression of cleaved caspase-3, cleaved casepase-9, and cleaved PARP. These results indicated that the suppression of protective autophagy by (GlcN)₅in HepG2 cells could further lead to apoptosis and inhibition on cell proliferation.

COS treatment suppressed the proliferation of HepG2 tumor cells through inducing apoptosis. Among the four kinds of singular DP COS, (GlcN)₅ exerted the best effect on suppressing proliferation and inducing apoptosis. Further investigation revealed that (GlcN)₅ could increase Bax expression and decrease Bcl-2 expression both in mRNA and protein level. Moreover, (GlcN)₅ reduced the mitochondrial membrane potential, and released cytochrome c, thus inducing apoptosis via intrinsic apoptosis pathway. Additionally, (GlcN)₅ could block the fusion of lysosomes and autophagosomes to impair autophagy flux, which further suppressed the proliferation but promoted apoptosis in HepG2 cells. Our findings indicated that (GlcN)₅ could be a potential therapeutic agent for HCC. However, studies on the inhibition effect of (GlcN)₅ on various tumor cells and in vivo studies should be conducted to further substantiation.

HCC, hepatocellular carcinoma; COS, chitooligosaccharides; DP, degree of polymerization; FBS, fetal bovine serum; DMEM, Dulbecco’s Modified Eagle Medium; caspase, cysteinyl aspartate specific proteinase; LC3B, microtubule-associated proteins 1A/1B light chain 3B; PARP, poly ADP-ribose polymerase; LAMP, lysosomal-associated membrane protein; 3-MA, 3-methyladenine; CQ, chloroquine; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; DMSO, dimethyl sulfoxide; HPLC, high performance liquid chromatography; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; NMR, nuclear magnetic resonance; FT-IR, Fourier transform–infrared; (GlcN)5, chitopentaose; IC50, calculated 50% inhibition concentration; FITC, fluorescein isothiocyanate isomer I; PI, propidium iodide; PBS, phosphate buffer saline; MMP, mitochondrial membrane potential; TEM, transmission election microscopy; SD, standard deviation; ANOVA, analysis of variance

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Data availability statements

All data generated or analyzed during this study are included in this published article.

Competing interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported by “Shu Guang” project of Shanghai Municipal Education Commission and Shanghai Education Development Foundation (15SG28), the China Postdoctoral Science Foundation (2017M621392), Fundamental Research Funds for the Central Universities (22221818014), the Open Project Funding of the State Key Laboratory of Bioreactor Engineering, ECUST (ZDXM2019), and the Shanghai PuJiang Program (18PJ1401900).

Author Contributions

Author Chunfeng Zhu curried out the assays, data collection, validation and drafted the manuscript. Author Liqiang Fan and Xuni Cao provided guidance on cell experiments. Author Quanming Xia and Jiachun Zhou provided comments on manuscript revision. Author Mengyao Zhao and Liming Zhao supervised the assays, data validation and worked in funding acquisition. All authors read and approved the final manuscript.

Acknowledgment

Not applicable.

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Chitopentaose Inhibits Hepatocellular Carcinoma by Inducing Mitochondrial Mediated Apoptosis and Suppressing Protective Autophagy

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