Yanping Zhang, Xiaojun Wang and Ping Cao*

Department of Gastroenterology, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, Shanxi Province 030032, China

*Corresponding Author:
Ping Cao
Department of Gastroenterology, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, Shanxi Province 030032, China
E-mail: success19830322@163.com
Date of Received 07 February 2022
Date of Revision 12 March 2023
Date of Acceptance 04 October 2023
Indian J Pharm Sci 2023;85(6):1755-1760  

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms

DOI: 10.36468/pharmaceutical-sciences.1238

Abstract

This study explored Sapindus saponins role in the proliferation and metastasis of colorectal cancer cells and its possible mechanism. SW620 cells were divided into 8 groups; control group, Sapindus saponin-low group, Sapindus saponin-middle group, Sapindus saponin-high group, microRNA+negative control group, microRNA- 5590-3p group, Sapindus saponin+anti-microRNA-negative control group and Sapindus saponin+anti-microRNA-5590-3p group. Cell proliferation and metastasis were determined using colony formation assay, wound healing assay, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay and transwell assay. MicroRNA-5590-3p expression was detected by quantitative reverse transcription-polymerase chain reaction. Matrix metalloproteinases-2 and matrix metalloproteinases-9 protein levels were examined by Western blot. SW620 cell proliferation inhibition rate and microRNA-5590-3p expression were increased, while colony numbers, invaded cell numbers, wound healing rate, matrix metalloproteinases-2 and matrix metalloproteinases-9 protein levels were decreased in Sapindus saponin-low group, Sapindus saponin-middle group, Sapindus saponin-high group in a dose-dependent manner. Besides, microRNA-5590-3p overexpression increased SW620 cell proliferation inhibition rate, while suppressed colony numbers, invaded cell numbers, wound healing rate, matrix metalloproteinases-2 and matrix metalloproteinases-9 protein levels. Compared with the Sapindus saponin+anti-microRNA-negative control group, SW620 cell proliferation inhibition rate was reduced, while colony numbers, invaded cell numbers, wound healing rate, matrix metalloproteinases-2 and matrix metalloproteinases-9 protein levels were enhanced in the Sapindus saponin+anti-microRNA-5590- 3p group. Sapindus saponin could repress colorectal cancer cell proliferation and metastasis by upregulating microRNA-5590-3p.

Keywords

Colorectal cancer, Sapindus saponin, microRNA-5590-3p, matrix metalloproteinases, chemotherapy

Colorectal Cancer (CRC) is a common clinical malignant tumor[1,2]. Chemotherapy drugs and other drugs have great toxic and side effects, and patients are prone to develop drug resistance leading to poor treatment effect[3,4]. It is of great significance to find drugs that can inhibit CRC cell proliferation and metastasis. Traditional Chinese Medicine (TCM) has anti-tumor effects and can regulate CRC cell biological behaviors[5,6]. It has been reported that Sapindus saponin inhibits proliferation in lung cancer and hepatocellular carcinoma[7,8]. However, the effect of Sapindus saponin on CRC progression is still unknown.

MicroRNA (miRNA) is widely involved in regulating human disease processes[9,10]. Studies had shown that miR-5590-3p downregulation facilitated proliferation in prostate cancer cells[11]. Besides, miR-5590-3p had been confirmed to restrain cell invasion in ovarian cancer and renal cancer[12,13]. Therefore, miR-5590-3p may serve as a tumor suppressor. However, its role in CRC progression has not been elucidated.

In this, we found that Sapindus saponin has a promotion effect on miR-5590-3p expression, but whether Sapindus saponin mediates CRC progression by increasing miR-5590-3p remains unclear. Basing on the above, we hypothesized that Sapindus saponin inhibited CRC cell functions by upregulating miR-5590-3p.

Materials and Methods

Cell culture and grouping:

SW620 cells (Procell, Wuhan, China) were grown in Dulbecco's Modified Eagle Medium (DMEM) plus 10 % Fetal Bovine Saline (FBS) and 1 % dualantibiotic (Invitrogen, Carlsbad, California, United States of America (USA)). Cells were treated with Sapindus saponin (Pushida, Wuhan, China) for 24 h[8], and recorded as Sapindus saponin+low (25 μg/ml) group, Sapindus saponin+middle (50 μg/ ml) group and Sapindus saponin+high (100 μg/ml) group. Normal cultured SW620 cells were used as control group. Lipofectamine 3000 (Invitrogen) was used for cell transfection. miR-5590-3p mimic and miR-NC were transfected into SW620 cells, recording as miR-NC group and miR-5590- 3p group. Besides, SW620 cells were transfected with anti-miR-5590-3p and anti-miR-NC followed by treated with 100 μg/ml Sapindus saponin for 24 h, recording as Sapindus saponin+anti-miR-NC group and Sapindus saponin+anti-miR-5590-3p group.

3-[4,5-Dimethylthiazol-2-yl]-2,5 Diphenyl Tetrazolium Bromide (MTT) assay:

SW620 cells were inoculated on 96-well plates and incubated with MTT solution (Beyotime, Shanghai, China) followed by treated with Dimethyl Sulfoxide (DMSO). Absorbance at 490 nm was detected by microplate reader to count cell proliferation inhibition rate.

Colony formation assay:

SW620 cells were cultured for 14 d in 6-well plates. After that, colonies were fixed and colony numbers were counted under a microscope.

Wound healing assay:

After reached 90 % confluences in 6-well plates, SW620 cells were scratched by a 200 μl pipette tip. Wound area was observed under a microscope to count wound healing rate.

Transwell assay:

SW620 cells were seeded into Matrigel-coated transwell upper chamber (Corning Inc., Corning, New York, USA), and complete medium were filled in lower chamber. Then, invaded cell numbers were counted under a microscope.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR):

Extracted Ribonucleic Acid (RNA) was reversetranscribed into complimentary Deoxyribonucleic Acid (cDNA), and PCR amplification was performed with SYBR Green (Takara, Dalian, China). miR-5590-3p expression was analyzed by 2−ΔΔCt method.

Wesern blot:

Protein extracted by Radioimmunoprecipitation Assay (RIPA) buffer was transferred onto Polyvinylidene Difluoride (PVDF) membranes after separated by Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel. Membrane was incubated with anti-Matrix Metalloproteinase (MMP)-2, anti-MMP-9 or anti-Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) followed by treated with secondary antibody. Protein signals were displayed using Enhanced Chemiluminescence (ECL) reagents (Beyotime).

Statistical analysis:

Measurement data were expressed as x̄ ±s, and analyzed by Statistical Package for the Social Sciences (SPSS) 20.0 software. Comparison was assessed by Student’s t-test or Analysis of Variance (ANOVA). p<0.05 was considered statistically significant.

Results and Discussion

Has showed in Table 1, cell proliferation inhibition rate was increased, while colony numbers were decreased in the Sapindus saponin-low, Sapindus saponin-middle, and Sapindus saponin-high groups in a dose-dependent manner.

Group Inhibition rate (%) Colony numbers
Control 0.00±0.00 111.70±10.26
Sapindus saponin+low 24.57±2.07* 85.38±7.95*
Sapindus saponin+middle 45.13±5.02*# 65.50±5.12#
Sapindus saponin+high 71.72±5.97*#& 48.38±4.43*#&
F 513.020 124.501
p 0.000 0.000

Table 1: Effect of Sapindus Saponin on Sw620 Cell Proliferation (X̄±S, N=9)

Moreover, MMP-2 and MMP-9 protein levels, wound healing rate and invaded cell numbers were reduced in the Sapindus saponin-low, Sapindus saponin-middle, and Sapindus saponin-high groups in a dose-dependent manner as shown in fig. 1 and Table 2. miR-5590-3p expression was elevated in the Sapindus saponin-low, Sapindus saponin-middle and Sapindus saponin-high groups in a dose-dependent manner as shown in Table 3.

pharmaceutical-sciences-meta

Fig. 1: Effect of Sapindus saponin on metastasis-related protein expression in SW620 cells Note: (Image): Control; (Image): Sapindus saponin-low; (Image): Sapindus saponin-middle and (Image): Sapindus saponin-high

Group Wound healing rate (%) Invaded cell numbers
Control 72.71±5.09 127.41±11.23
Sapindus saponin+low 55.81±4.69* 93.26±7.02*
Sapindus saponin+middle 42.39±3.43*# 73.43±6.66#
Sapindus saponin+high 25.61±2.05*#& 54.85±8.25*#&
F 225.313 120.483
p 0.000 0.000

Table 2: Effect of Sapindus Saponin on Sw620 Cell Migration and Invasion (X̄±S, N=9)

Group miR-5590-3p
Control 1.00±0.01
Sapindus saponin+low 1.62±0.13*
Sapindus saponin+middle 2.41±0.21*#
Sapindus saponin+high 3.11±0.26*#&
F 236.986
p 0.000

Table 3: Effect of Sapindus Saponin on Mir-5590-3p Expression in Sw620 Cells (X̄±S, N=9)

miR-5590-3p mimic was used to overexpress miR-5590-3p expression in SW620 cells. Besides, miR-5590-3p overexpression decreased colony numbers, invaded cell numbers, wound healing rate, MMP-2 and MMP-9 protein levels, while elevated cell proliferation inhibition rate as shown in fig. 2 and Table 4.

pharmaceutical-sciences-protein

Fig. 2: miR-5590-3p overexpression regulated metastasis-related protein expression in SW620 cells Note: (Image): miR-NC and ( Image): miR-5590-3p

Group miR-5590-3p Inhibition rate (%) Colony numbers Wound healing rate (%) Invaded cell numbers
miR-NC 1.00±0.00 7.38±0.57 113.07±12.13 73.15±6.35 129.16±10.13
miR-5590-3p 3.02±0.27* 25.34±2.08* 53.76±4.62* 32.37±3.45* 60.68±4.96*
t 22.444 24.983 13.708 16.929 18.214
p 0.000 0.000 0.000 0.000 0.000

Table 4: miR-5590-3p Overexpression Regulated Sw620 Cell Proliferation and Metastasis (x̄±s, n=9)

Meanwhile, anti-miR-5590-3p was used to reduce miR-5590-3p expression in Sapindus saponintreated SW620 cells. Functional experiments showed that colony numbers, invaded cell numbers, wound healing rate, MMP-2 and MMP-9 protein levels were enhanced, while cell proliferation inhibition rate was reduced in Sapindus saponin+anti-miR-5590-3p group as shown in fig. 3 and Table 5.

pharmaceutical-sciences-reverse

Fig. 3: miR-5590-3p inhibitor reversed Sapindus saponin-mediated metastasis-related protein expression in SW620 cells Note: (Image): Sapindus saponin+anti-miR-NC and (Image): Sapindus saponin+anti-miR-5590-3p

Group miR-5590-3p Inhibition rate (%) Colony numbers Wound healing rate (%) Invaded cell numbers
Sapindus saponin+anti-miR-NC 1.00±0.00 72.18±5.62 46.52±4.51 23.53±2.11 53.82±4.69
Sapindus saponin+anti-miR-5590-3p 0.43±0.04* 27.17±2.69* 89.28±6.58* 60.74±4.84* 106.86±11.85*
t 42.75 21.672 16.081 21.142 12.486
p 0.000 0.000 0.000 0.000 0.000

Table 5: miR-5590-3p Inhibitor Reversed Sapindus Saponin-Mediated Sw620 Cell Proliferation and Metastasis (x̄±s, n=9)

TCM may exert anti-CRC effects by regulating multiple signaling pathways or gene expression[14,15]. miRNAs are aberrantly expressed in CRC tissues and can affect CRC cell biological behaviors[16,17]. However, whether miRNAs can be a potential target for CRC treatment with TCM need to be further explored.

Sapindus saponin plays an important role in many cancers. Sapindus saponin could facilitate apoptosis in lung cancer cells[7]. Also, Sapindus saponin was found to inhibit proliferation in hepatocellular carcinoma cells[8]. However, Sapindus saponins role in CRC has been reported relatively little. Our data showed that SW620 cell proliferation was markedly reduced after treatment with Sapindus saponin, suggesting that Sapindus saponin could suppress CRC cell proliferation. MMP-2 and MMP-9 belong to matrix metalloproteinases, whose expression are up-regulated in CRC and can facilitate cell metastasis[18,19]. Here, Sapindus saponin was found to reduce wound healing rate, invaded cell numbers, MMP-2 and MMP-9 levels in SW620 cells, indicating that Sapindus saponin inhibited CRC cell metastasis.

miR-5590-3p is aberrantly expressed in many cancers[11,20]. miR-5590-3p overexpression suppressed proliferation in breast cancer[21]. miR- 5590-3p upregulation inhibited renal carcinoma cell metastasis[22]. In this study, miR-5590-3p was upregulated in Sapindus saponin-treated SW620 cells. Meanwhile, miR-5590-3p overexpression reduced SW620 cell proliferation and metastasis, whereas its inhibition also reversed the effect of Sapindus saponin on SW620 cell functions, indicating that Sapindus saponin repressed CRC cell progression by promoting miR-5590-3p expression.

In conclusion, Sapindus saponin suppressed CRC cell proliferation and metastasis by upregulating miR-5590-3p. However, whether Sapindus saponin can exert anti-cancer effects by regulating the other genes expression or signaling pathways needs to be further investigated.

Conflict of interests:

The authors declared no conflict of interests.

References