Chitooligosaccharide Inhabits Migration of Pancreatic Cancer Cells by Regulating Epithelial– Mesenchymal Transition

doi:10.21203/rs.3.rs-4303848/v1

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Chitooligosaccharide Inhabits Migration of Pancreatic Cancer Cells by Regulating Epithelial– Mesenchymal Transition

https://doi.org/10.21203/rs.3.rs-4303848/v1

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Background

Studies have confirmed that chito-oligosaccharide (COS) has antitumour effect, but research on its effect on pancreatic cancer (PC) remains limited and unclear. This study aimed to explore effects of COS on PC cells.

Method

We used different concentrations of COS to treat PC cells and conducted Cell Counting Kit-8, wound-healing, and transwell assays to evaluate the proliferation, invasion, and migration ability of PC cells, respectively. Western blotting was conducted to assess the expression levels of epithelial–mesenchymal transition (EMT) related markers.

Result

The proliferation, invasion, and migration ability of PC cells (PANC-1 and MIAPaCa-2) gradually decreased in a manner dependent on COS concentration. COS at 10 mg/mL exerted the strongest inhibitory effect on the two PC cell lines. At 10 mg/mL, the proliferative activity was 60.61% ± 5.25% and 64.02% ± 4.96%, respectively; the invasive ability was (18.67 ± 4.416) and (31.33 ± 3.162), respectively; and the cell-migration ability was 26.83% ± 0.442% and 17.66% ± 0.647%, respectively. The expression levels of N-cadherin and vimentin were significantly downregulated in PANC-1 cells (0.198 ± 0.047 and 0.225 ± 0.038, respectively) and MIAPaCa-2 cells (0.214 ± 0.094 and 0.214 ± 0.094, respectively) at 10 mg/mL, respectively. Conversely, E-cadherin was upregulated (0.460 ± 0.037 and 0.491 ± 0.047, respectively). Compared with control group, the differences were statistically significant.

Conclusion

The upregulation of E-cadherin and the downregulation of vimentin and N-cadherin suggested that the specific mechanism of COS in PC may be related to EMT. This study provided a new direction for PC treatment.

Chito-oligosaccharide

pancreatic cancer

epithelial–mesenchymal transition

In recent years, the morbidity of pancreatic cancer (PC) has increased significantly in China and other countries. In accordance with the 2021 statistics from the National Cancer Center of China, PC ranks 7th in the incidence of malignant tumours amongst males and 11th amongst females, and 6th in mortality rate related to malignant tumours[1].

With the increase in the incidence rate of PC, research on its treatment has also gradually increased. However, the physical and chemical properties of some drugs affect the efficiency of their absorption and utilisation. The side effects of drugs also limit their application. Therefore, choosing drugs or carriers that are relatively safe, effective and highly bioavailable is important. Studies have shown that a drug that can selectively cause damage to tumour cells with no toxicity on normal diploid cells is considered an ideal anticancer agent. Chitosan oligosaccharide (COS) is a promising drug candidate and food ingredient, because they are naturally biocompatible, nontoxic and nonallergenic to living tissues[2]. COS is composed of β-(1, 4) glucosamine connected by glycosidic bond. It exhibits complete water solubility, low molecular weight (Mw), high degree of polymerisation (DP), high degree of deacetylation (DD) and less viscosity[3], endowing it with important biological characteristics. In particular, COS and its derivatives have been proven to exhibit various biological activities[4], including anti-inflammatory, antitumour[5–7], immune stimulation[8] and the promotion of tissue regeneration[9]. Some studies have also confirmed that COS can interfere with cancer cell proliferation at different stages of growth, invasion and metastasis[10]. In vitro studies have shown that COS can induce the death of some important cancer cells. In the study of specific tumour cells, the average half maximal inhibitory concentration (IC₅₀) of COS for inducing cytotoxicity is between 25 µg/mL and 50 µg/mL, depending on tumour types[3].

Therefore, in accordance with previous experimental results, the current study selected different concentrations of COS (0.625, 2.5 and 10 mg/mL) to treat PC cells (PANC-1 and MIAPaCa-2), explore the changes in the expression of epithelial–mesenchymal transition (EMT) molecular markers [epithelial cadherin (E-cadherin), neural cadherin (N-cadherin) and vimentin] in PC cells after treatment with COS and further investigate how COS inhibits the proliferation, invasion and migration of PC cells.

Materials

PC cells (PANC-1 and MIAPaCa-2) were provided by Jiangsu Kaiji Biotechnology Co., Ltd. The parameters of the EMT markers used were as follows: E-cadherin (Abcam ab40772; Mw: 80–120 kd, dilution ratio: 1:10000), N-cadherin (Abcam ab98952; Mw: 100 kd, dilution ratio: 1:500) and vimentin (Abcam ab92547; Mw: 54 kd, dilution ratio: 1:1000). The concentration of the protein electrophoresis gel was 10%. The following were purchased from Jiangsu Kaiji Biotechnology Co., Ltd. (China): Cell Counting Kit-8, incomplete Dulbecco’s Modified Eagle’s Medium (DMEM) with high sugar and medium sugar (including double antibody), incomplete DMEM/F12 medium (including double antibody), whole protein extraction kit, bicinchoninic acid (BCA) protein content detection kit, sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gel preparation kit, pre-staining protein Mw, 5 × SDS–PAGE protein loading buffer, 1 × Tris-glycine protein electrophoresis buffer, Western blot detection kit and the developing and fixing reagents. Meanwhile, 96-well cell culture plate, 24-well cell culture plate, transwell cell and 6-well cell culture plate were bought from Corning Incorporated (USA). Crystal violet was procured from Sigma C3886 (USA).

Methods

CCK-8 assay determines the effect of different COS on cell proliferation ability

The cultured cells were digested, counted and configured to cell suspensions with a concentration of 4 × 10⁴/mL. The cells were resuspended, and 100 µL samples were taken from each well, inoculated into the 96-well plate and incubated at 37 ℃ in a 5% CO₂ incubator for 24 h. The old culture medium was discarded, and 100 µL of COS media with concentrations of 0.625, 2.5 and 10 mg/mL were added to each well in the PANC-1 group. Similarly, 100 µL of COS media with concentrations of 0.625, 1.25 and 2.5 mg/mL were added to each well in the MIAPaCa-2 group. Both groups were incubated for 72 h, whilst a negative control group was established. Then, 10 µL of CCK-8 detection solution was added to each well and incubated for 2 h. Subsequently, enzyme-linked immunosorbent assay was performed at an optical density of 450 nm. Three wells were established in each group to calculate cell proliferation ability (i.e. cell viability).

Transwell invasion assay determines the effects of different COS concentrations on cell invasion ability

Transwell invasion assay was conducted to assess the invasive capacity of cells. The transwell cell chamber was purchased from Corning Incorporated (USA). PANC-1 and MIAPaCa-2 cells were configured with COS (0.625, 2.5 and 10 mg/mL) into 1 × 10⁵/mL cell suspensions. Then, 100 µL of the cell suspension was added to the upper cell chamber, whilst 500 µL of the culture medium with fetal bovine serum was added to the lower chamber. The suspension and medium were incubated at 37 ℃ in a 5% CO₂ incubator for 24 h. Thereafter, the matrix adhesive and the cells in the upper chamber were wiped off with a cotton swab. The transwell was removed, inverted and air-dried. The chamber was placed in the 24-well plate with 500 µL of 0.1% crystal violet. The membrane was immersed in the dye at 37 ℃ for 30 min. After cleaning with phosphate-buffered saline (PBS), three fields of view on the diameter of the biological inverted microscope were randomly taken, magnified at 200×. The number of cells was then counted to evaluate their invasiveness.

Wound healing assay determines the effects of different COS concentrations on cell migration ability

Wound healing assay was conducted to evaluate the migration ability of tumour cells. PANC-1 and MIAPaCa-2 cells of the logarithmic growth phase were digested and inoculated into the six-well plate. The following day, when cell aggregation reached about 80%, a sterile pipette tip was used to scratch the surface of a monolayer. The floating cells were washed with PBS and then replaced in fresh culture medium with COS (0.625, 2.5 and 10 mg/mL). The cells were subsequently placed in a cell incubator for 24 h for further cultivation. Then, photographs were taken using a biological inverted microscope (magnification: 100×) to observe cell migration. The distance of cell migration was measured with the following formula: cell migration rate = (0 h migration distance − 24 h migration distance)/0 h migration distance × 100%.

Western blot analysis detects the expression levels of E-cadherin, N-cadherin and vimentin proteins at different COS concentrations

After treatment with COS (0.625, 2.5 and 10 mg/mL), the cells were collected and a cold lysis buffer solution was used to extract the total cell proteins. Protein concentration was measured using BCA assay. Firstly, 50 µg of relevant samples were added per well, separated with 10% SDS–PAGE and transferred to a polyvinylidene difluoride membrane. Secondly, the membrane was blocked in 5% skimmed milk at room temperature and shaken for 2 h. Thirdly, the membrane was placed in a plate that contained the primary antibody (diluted with Western first antibody diluent) and incubated overnight at 4 ℃ on a shaking table. The following day, the membrane was shaken at room temperature for 30 min. The first antibody was discarded, and the membrane was cleaned with TBST. The second antibody was then diluted with a diluent, shaken at room temperature for 2 h and recovered after the reaction. The membrane was washed with TBST. The proteins were visualised by using an enhanced chemiluminescence system, and the gel imaging system SYNGENE G: BOXChemiXR5 (UK) was used to detect the luminous signal. The protein bands were scanned and quantified on Image J software with glyceraldehyde 3-phosphate dehydrogenase for normalisation.

Statistical analyses

Data were presented as mean values ± standard deviation and analysed using SPSS 25.0 software (SPSS, Inc., Chicago, IL, USA). One-way ANOVA with LSD-t tests was performed to compare statistical significance in various groups. Each group of experiments should be independently repeated at least three times. P < 0.05 was considered a statistically significant difference.

COS inhibits the proliferation activity of PC cells

To observe the effect of COS on the proliferation activity of PANC-1 and MIAPaCa-2 cells, CCK-8 assay was conducted to detect the proliferation activity of cells treated with different COS concentrations for 72 h. The results showed that COS can inhibit the proliferation activity of cells. After intervention with 0.625, 2.5 and 10 mg/mL of COS, the proliferation activity of PANC-1 cells was (97.53 ± 6.35)%, (85.36 ± 3.67)% and (60.61 ± 5.25)%, respectively. After intervention with 0.625, 1.25 and 2.5 mg/mL of COS, the proliferation activity of MIAPaCa-2 cells was (93.07 ± 1.97)%, (89.65 ± 2.22)% and (64.02 ± 4.96)%, respectively. The proliferation activity of PANC-1 and MIAPaCa-2 cells gradually decreased in a concentration-dependent manner (PANC-1: F = 47.888, P = 0.000; MIAPaCa-2: F = 88.476, P = 0.000), as shown in Fig. 1.

Different COS concentrations influence the invasion and migration abilities of PC cells

The transwell invasion assay results demonstrated the invasiveness of cells (Figs. 2–3). After treatment with 0.625, 2.5 and 10 mg/mL of COS, transmembrane cells per high-power field of view in the PANC-1 group were (185.22 ± 1.986), (137.00 ± 2.598) and (18.67 ± 4.416), respectively. In the MIAPaCa-2 group, the values were (164.44 ± 2.698), (104.89 ± 2.977) and (31.33 ± 3.162), respectively. The invasive ability of the two groups gradually decreased in a concentration-dependent manner, and the difference was statistically significant (PANC-1: F = 8764.532, P = 0 000; MIAPaCa-2: F = 8764.532, P = 0.000).

The healing of the scratched area after 24 h of cell culture can reflect the relative migration ability of cells (Figs. 4 and 5). When COS concentration was 0.625, 2.5 and 10 mg/mL, the scratch-healing rates of the PANC-1 group were (84.27 ± 0.819)%, (72.31 ± 0.685)% and (26.83 ± 0.442)%, respectively. Meanwhile, those of the MIAPaCa-2 group were (64.27 ± 0.548)%, (41.19 ± 0.690)% and (17.66 ± 0.647)%, respectively. As COS concentration increased, cell migration ability gradually decreased. The difference was statistically significant (PANC-1: F = 15758.529, P = 0.000; MIAPaCa-2: F = 10841.293, P = 0.000). COS exerts a concentration-dependent effect on inhibiting the migration and invasion of PC cells.

Comparison of E-cadherin, N-cadherin and vimentin protein expression levels in different COS concentrations

With an increase in COS concentration, the expression levels of N-cadherin and vimentin was significantly decreased in the PANC-1 and MIAPaCa-2 groups, whereas the expression of E-cadherin was increased (Table 1, Figs. 6 and Fig. 7).

Table 1

Comparison of relative expression levels of E-cadherin, N-cadherin, and Vimentin proteins in each group(x ± s)
Group	COS concentration (mg/ml)	E-cadherin	N-cadherin	Vimentin
PANC−1	0.625	0.246 ± 0.036	0.500 ± 0.168	0.505 ± 0.148
	2.500	0.343 ± 0.057*	0.267 ± 0.085*	0.337 ± 0.068*
	10.000	0.460 ± 0.037*	0.198 ± 0.047*	0.225 ± 0.038*
MIAPaCa−2	0.625	0.272 ± 0.036	0.450 ± 0.164	0.465 ± 0.064
	2.500	0.351 ± 0.062#	0.313 ± 0.129	0.370 ± 0.086#
	10.000	0.491 ± 0.047#	0.214 ± 0.094#	0.214 ± 0.094#

compared with the control group, PANC-1 * P < 0.05;MIAPaCa-2 # P < 0.05

In the current study, the effect of COS on PC cells was investigated. Through CCK-8, transwell and wound-healing assays, we determined that the proliferation, migration and invasion of PANC-1 and MIAPaCa-2 cells were gradually reduced with an increase in COS concentration. Simultaneously, the expression of E-cadherin was increased, whereas the expression levels of vimentin and N-cadherin were decreased when the related cell markers of PC were detected. The COS concentration of 10 mg/mL exhibited the strongest inhibitory effect on the two PC cell lines.

PC is a common clinical malignant tumour that occurs mostly amongst men. In recent years, its morbidity has presented an increasing trend, with hidden symptoms and poor specificity. This disease develops rapidly, is difficult to diagnose early and has a high mortality rate[10, 11]. However, only a few studies have been conducted on COS and PC. In the current work, we conducted the first study on the effect of different COS concentrations on PC cells and found that COS inhibited the proliferation, migration and invasion of PC cells in a concentration-dependent manner (Fig. 1.2.4). Studies on other tumour cells have also confirmed the ability of COS to inhibit cell migration and invasion.

Shen et al. investigated the antitumour and antimetastatic potentials, and the pathways affected by COS in the human hepatocellular carcinoma cell line, HepG2, which was extracted from fungi. They discovered that in vitro COS significantly inhibited cell proliferation, and HepG2 cells were the most sensitive cancer cells to COS compared with stomach adenocarcinoma AGS cells and colon adenocarcinoma COLO 205 cells. Their data showed that COS treatment resulted in a concentration-dependent inhibition of cell growth in HepG2 cells[12]. Pan et al used low-degree-polymerised (DP = 2–6) COS materials. In vitro assays showed that COS elicited antitumour activity against osteosarcoma cells. They found that COS exerted significant effects on cell growth, metastasis inhibition, apoptosis and autophagy induction[13]. Jiang et al. found that in Lewis lung carcinoma, human non-small-cell lung cancer A549 cells exhibited an unusual arrangement after being incubated with various COS concentrations for 48 h, with reduced cell attachment numbers compared with that in the control group[14]. Our research results further confirmed this effect on PC, providing a richer experimental and theoretical basis for the clinical application of COS.

In relevant EMT research[15], the appearance of EMT leads to high tumour metastasis, poor prognosis and extremely low survival rate[16]. The epithelial cells of PC acquire the mesenchymal phenotype via EMT to increase anti-apoptosis, migration and invasion abilities[17]. For cancer stem cells, which can be isolated from solid malignancies, such as PC, Scheel et al. found that the expression of EMT markers exhibited corresponding changes. The expression of the epithelial marker protein E-cadherin was significantly up-regulated, whilst the expression levels of mesenchymal marker proteins, such as N-cadherin, vimentin and fibronectin, were down-regulated[18]. Therefore, we firstly explored the effect of different COS concentrations on the expression of marker proteins in epithelial and mesenchymal cells during EMT in PC cells. Through Western blot, we found that the expression of the epithelial cell marker E-cadherin in PANC-1 and MIAPaCa-2 cells increased with an increase in COS concentration, whereas the expression levels of the mesenchymal cell markers vimentin and N-cadherin were inhibited. In PC tissue, the deficiency of E-cadherin is positively correlated with the differentiation of PC and lymph node metastasis. Increased vimentin or fibronectin and decreased E-cadherin are correlated with high metastatic potential[19] and poor survival[20]. Except for E-cadherin, Hong et al. found that pancreatic tumour tissues exhibited higher than threefold expression of total vimentin protein than in the analysed lung, colon and ovarian tumours[21]. Accumulating evidence has shown that the elevated expression of N-cadherin is related to tumour aggressiveness and that N-cadherin is positively expressed in many types of tumours, such as pancreatic carcinoma[22, 23]. Shintani et al. found that N-cadherin expression increased the invasive and metastatic capacities of BxPC-3 cells in an orthotopic mouse model for PC[24]. Therefore, we speculate that COS can increase the expression of E-cadherin in a concentration-dependent manner, inhibit the expression levels of vimentin and N-cadherin, and finally, inhibit EMT. These processes may be the mechanism behind its inhibition of PC cell migration and invasion. Studies have found that COS may inhibit colon cancer cells by regulating the body’s immune response (colon 26-bearing mouse model)[25]. However, COS has not been found to affect the occurrence and metastasis of PC by regulating EMT. Simultaneously, no studies have been conducted on the expression levels of COS and E-cadherin, N-cadherin and vimentin proteins. Therefore, our study is the first to determine that COS inhibits EMT in PC.

we found that COS inhibits the proliferation, migration and invasion of PC cells in a concentration-dependent manner. We speculated that this phenomenon is related to its inhibition of EMT, providing a new basis for the application of COS in the treatment of PC. However, we only studied the effect of different COS concentrations on PC cells in this experiment and did not analyse the different action times, which may require further research. Simultaneously, this experiment only preliminarily studied PC cells in vitro. Compared with the complexity of the body’s internal environment, establishing an animal model of tumour xenotransplantation may be necessary to further study the effect of COS on PC in vivo.

This work was supported by the Science Foundation of Liaoning (grant no. 2019-MS-352) and the Science and Technology Foundation of Shenyang (grant no. 19-112-4-046).

Contributions:

Xu Liu proposed the study and performed research and wrote the first draft. Xu Liu, and Han Liu collected and analyzed the data. All authors contributed to the design and interpretation of the study and the final approval of manuscript。

This work was supported by the Science Foundation of Liaoning (grant no. 2019-MS-352) and the Science and Technology Foundation of Shenyang (grant no. 19-112-4-046).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form. The authors have no conflicts of interest to declare.

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Chitooligosaccharide Inhabits Migration of Pancreatic Cancer Cells by Regulating Epithelial– Mesenchymal Transition

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