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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 8  |  Page : 232-236

Solanum nigrum polysaccharide inhibits tumor growth in H22-bearing mice through regulation of caspase-3 and bcl-2


1 Department of Pharmacy, Jiaxing University College of Medicine, Jiaxing 314001, China
2 Department of Pathology, Zhejiang Provincial Corps Hospital, Chinese People's Armed Police Force, Jiaxing 314000, China

Date of Web Publication26-Mar-2018

Correspondence Address:
Yueyan Huang
Jiaxing University, No. 118 Jiahang Road, Jiaxing City, Zhejiang Province
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.206862

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 > Abstract 


Objective: The objective of this study was to investigate the effect of Solanum nigrum polysaccharides (SNPs) on tumor growth in H22 hepatocarcinoma cells bearing mice and explore the mechanism by focusing on the regulation of the expression of caspase-3 and bcl-2.
Materials and Methods: Totally, 50 mice bearing with H22 cells were randomly divided into five groups: Model group, cyclophosphamide group (CTX, 30 mg/kg), SNP groups with low, medium, and high doses of SNP (30, 60, and 120 mg/kg). Twenty-four hours after inoculation of H22 cells, CTX or SNP were given by gavage once a day for 10 days. The growth of tumor was observed. The tumor inhibition rate, indexes of the spleen and thymus were calculated. The immunohistochemical method was used for the determination of caspase-3 and bcl-2 expression in the tumor tissue.
Results: SNP (30, 60, and 120 mg/kg) reduced the average tumor weight compared with that in model group in a dose-dependent manner, and the tumor inhibition rates were 37.73%, 38.24%, and 42.60%, respectively. In addition, SNP dose dependently increased the index of the thymus compared with that of the CTX group. Immunohistochemistry results showed that the protein expression of caspase-3 in SNP groups was higher, but the expression of bcl-2 was lower than that in model group in a dose-dependent manner.
Conclusion: SNP inhibited the growth of tumor in H22-bearing mice and protected the immune organ. The mechanism underlying the inhibition of tumor might be related to the upregulation of caspase-3 and downregulation of bcl-2.

Keywords: Antitumor, bcl-2, caspase-3, H22 hepatocarcinoma cells, Solanum nigrum polysaccharide


How to cite this article:
Huang Y, Yin M, Pan L, Yu Q, Zhu Q, Xu W, Ding B, Ji Y, Zhou J. Solanum nigrum polysaccharide inhibits tumor growth in H22-bearing mice through regulation of caspase-3 and bcl-2. J Can Res Ther 2018;14, Suppl S1:232-6

How to cite this URL:
Huang Y, Yin M, Pan L, Yu Q, Zhu Q, Xu W, Ding B, Ji Y, Zhou J. Solanum nigrum polysaccharide inhibits tumor growth in H22-bearing mice through regulation of caspase-3 and bcl-2. J Can Res Ther [serial online] 2018 [cited 2019 Sep 15];14:232-6. Available from: http://www.cancerjournal.net/text.asp?2018/14/8/232/206862




 > Introduction Top


The imbalance between cell proliferation and apoptosis is an important mechanism of the occurrence and development of the malignant tumor.[1] Caspase-3, one of the important members in the cysteine protease (caspase) family, is the key regulator of mitochondrial apoptosis.[2],[3] Bcl-2 is also the key regulator in mitochondrial apoptosis and is closely associated with mammalian cell apoptosis.[4] The overexpression of bcl-2 inhibits the apoptosis and delays the cell death. Therefore, effective regulation of caspase-3 and bcl-2 might be using for antitumors.

Solanum nigrum L., one of the traditional Chinese medicines (TCMs), possesses the properties of relieving heat, reducing toxicity, inducing diuresis, and eliminating edema.[5],[6]S. nigrum contains steroidal alkaloids, polysaccharides, saponins, and other ingredients. Studies from pharmacological and clinical researches found that S. nigrum possesses obvious cytotoxicity to the tumor and inhibits tumor cell growth mainly through the induction of apoptosis, which mainly depends on its alkaloid and polysaccharide compositions. S. nigrum polysaccharides (SNPs) are the main components in S. nigrum. The previous study focused on water-soluble polysaccharide crude extract which exhibited the antitumor activity.[7],[8] However, the mechanism of its antitumor effect is not clear. We obtained highly refined SNP using the potent method. In this experiment, H22 hepatocarcinoma cells bearing mice were used to observe the effect of SNP on tumor growth and explore the mechanism by focusing on the regulation of the expression of caspase-3 and bcl-2. This study will provide a scientific basis for developing less toxic and more effective antitumor new drug.


 > Materials and Methods Top


Drugs and reagents

SNP was prepared in our laboratory with the purity of more than 98%. The delicate purification processing method was given the patent application number of 201510534404.3. Briefly, SNP was obtained from S. nigrum raw material by flash extraction, concentration, alcohol precipitation, and drying, followed by removal of protein, decoloring, and alcohol precipitation. The content of polysaccharide was determined using ultraviolet spectrophotometer based on the method of phenol-sulfuric acid. Cyclophosphamide (CTX) injection was produced by Jiangsu Hengrui Medicine Co., Ltd., China. RPMI1640 medium and phosphate buffer solution were purchased from Gino Biopharmaceutical Technology Co., Ltd. Hangzhou, China. Newborn bovine serum was from Zhejiang Tianhang Biological Technology Co., Ltd., China. MTT, citric antigen retrieval buffers, DAB color reagent kit, and mouse monoclonal antibodies caspase-3 and bcl-2 immunohistochemical kit were purchased from Guangzhou Shenda Biological Products Co., Ltd. Guangzhou, China. Formaldehyde, ethanol and xylene, dimethyl sulfoxide, and other reagents are domestic analytical pure.

Animals and cell lines

Specific pathogen-free ICR mice (half male and half female) with the weight of 22–26 g were purchased from the Animal Center of Zhejiang University of Traditional Chinese Medicine, China (Certificate Number: No. scxk [Zhejiang] 2014-0001). Murine H22 hepatocarcinoma cell lines were provided by the Experimental Animal Center of Zhejiang University of Traditional Chinese Medicine.

Preparation of Solanum nigrum polysaccharide solution

SNP was added into distilled water and ultrasonically vibrated for 20 min until totally dissolved. The final concentrations of SNP are 2, 4, and 8 mg/ml, respectively.

H22 cells bearing mice model and groups

H22 cell lines with a concentration of 107 cells/ml were intraperitoneally injected to mice. Seven days after the injection, the ascites of the H22-bearing mice were drawn out under aseptic conditions followed by being diluted with aseptic saline to the final concentration of 107 cells/ml. Trypan blue dying showed 98% of the living cells. The cells were subcutaneously inoculated into the right armpit with 0.2 ml per mouse to establish the H22 mice model. Twenty-four hours after inoculation, mice were weighed and randomized into five groups with ten rats in each group: model group in which mice were given saline by gavage; CTX group in which mice were given CTX by gavage; SNP groups in which mice were given SNP (30, 60, and 120 mg/kg/day) by gavage for 10 days, respectively.

Animal experiments

At the end of the experiments, the mice were sacrificed. The tumor tissues from the right armpit, thymus, and spleen were separated, respectively, and accurately weighed. The tumor inhibition rate and the indexes of the thymus and spleen were calculated using the following formula:

Tumor inhibition rate = (1 – [tumor weight in CTX or SNP group/tumor weight in model group]) × 100%.

Spleen (thymus) index = Spleen (thymus) weight (mg)/body weight (g).

Pathological changes of tumor tissues

Tumor tissues were fixed in 10% neutral formalin followed by dehydration, paraffin-embedded, cutting into slice with 4 μm of thickness and hematoxylin and eosin (H and E) staining. The pathological changes were observed under BX50 type microscope (Olympus).

Immunohistochemical analysis of caspase-3 and bcl-2 protein expression in tumor tissue

The immunohistochemical assay was performed as described previously. The paraffin-embedded tumor tissue was cut into slice with 4 μm of thickness followed by 3% H2O2 block, antigen repair with citric acid salt antigen retrieval buffers, addition of the first and second antibody, visualization using DAB, hematoxylin counterstain. According to the staining intensity and staining area, the positive staining was evaluated by two experimenters in a double-blind manner. The positive staining was defined as the existence of yellow, brown-yellow granules in the cell membrane or cytoplasm. The positive staining of five fields in each slice was analyzed. The percentage of positive staining was calculated in 200 tumor cells. According to [Table 1], the score was obtained by the addition of positive cell percentage score and the score of staining intensity.
Table 1: Evaluation of immunohistochemical detection

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Statistical analysis

Results are presented as mean ± standard deviation for each group. Statistical analysis was performed by one-way ANOVA and the Student-Newman-Keuls test using SPSS 16.0 (IBM, USA) software; P < 0.05 was considered statistically significant.


 > Results Top


Effect of Solanum nigrum polysaccharide on body weight and tumor weight in H22-bearing mice

As shown in [Figure 1], SNP dose dependently reduced the tumor weight in H22-bearing mice compared with model control. The inhibition rates were 37.73%, 38.24%, and 42.60%, respectively, from low to high doses of SNP. In addition, CTX inhibited the tumor weight by 68.25%. However, both CTX and SNP had no effects on body weight.
Figure 1: Effect of Solanum nigrum polysaccharide on tumor weight in H22-bearing mice. Fifty mice bearing with H22 cells were randomly divided into five groups: model group, cyclophosphamide group (30 mg/kg), and Solanum nigrum polysaccharide groups with low, medium, and high doses of Solanum nigrum polysaccharide (30, 60, and 120 mg/kg). (a) Representative photograph of tumor tissue. (b) Tumor weight (g). (c) inhibition rate (%). Data are expressed as the mean ± standard error mean n = 10; *P < 0.05 was considered statistically significant compared with model group

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Effect of Solanum nigrum polysaccharide on indexes of spleen and thymus on H22-bearing mice

The results showed that the indexes of spleen and thymus in CTX group were decreased compared with that in the model group; however, no statistical significance was found. SNP had no affection on the index of the spleen. However, SNP with low and high doses increased the index of thymus compared with CTX group [Figure 2].
Figure 2: Effect of Solanum nigrum polysaccharide on indexes of spleen and thymus on H22-bearing mice. Fifty mice bearing with H22 cells were randomly divided into five groups: model group, cyclophosphamide group (30 mg/kg), and Solanum nigrum polysaccharide groups with low, medium, and high doses of Solanum nigrum polysaccharide (30, 60, and 120 mg/kg). (a) Index of the spleen. (b) Index of the thymus. Data are expressed as the mean ± standard error mean n = 10; *P < 0.05 was considered statistically significant compared with cyclophosphamide

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Effect of Solanum nigrum polysaccharide on pathological changes in hematoxylin and eosin staining

H and E staining for pathological observation showed a plenty of tumor cells and prominent nucleoli [Figure 3]a and the infiltration into adipose tissue of tumor cells [Figure 3]b in model group. In CTX group, patchy necrosis [Figure 3]c, the invasion into the muscle and fat and necrosis of tumor cells [Figure 3]d were seen. However, infiltration into fat and extensive necrosis [Figure 3]e and apoptosis [Figure 3]f of tumor cells was found in the SNP (L) group. In addition, shrinking necrosis [Figure 3]g of tumor cells and thymic cell proliferation [Figure 3]h were found in the SNP (M) group.
Figure 3: Effect of Solanum nigrum polysaccharide on pathological changes in H and E staining. Forty mice bearing with H22 cells were randomly divided into four groups: model group, cyclophosphamide group (30 mg/kg), and Solanum nigrum polysaccharide groups with low and medium doses of Solanum nigrum polysaccharide (30, 60 mg/kg). A plenty of tumor cells and prominent nucleoli (a, ×400) and the infiltration into adipose tissue of tumor cells (b, ×200) in model group. In cyclophosphamide group, patchy necrosis (c, ×200), the invasion into the muscle and fat and necrosis of tumor cells (d, ×200) were seen. Infiltration into fat and extensive necrosis (e, ×100) and apoptosis (f, ×200) of tumor cells was found in the Solanum nigrum polysaccharide (L) group. In addition, shrinking necrosis (g, ×200) of tumor cells and thymic cell proliferation (h, ×200) were found in the Solanum nigrum polysaccharide (M) group

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Effect of Solanum nigrum polysaccharide on protein expression of caspase-3 and bcl-2 in tumor tissue

To investigate the mechanism underlying the inhibition of tumor by SNP, we determined whether SNP affects the apoptosis of tumor cells by testing the protein expression of caspase-3 and bcl-2, an important inhibitor of apoptosis protein, in tumor tissue. The results showed that SNP increased the protein expression of caspase-3 while decreased the expression of bcl-2 in tumor tissue compared with the model group in a dose-dependent manner, suggesting that the regulation of apoptosis-related genes and induction of apoptosis of tumor cells might contribute the inhibition of tumor by SNP [Figure 4].
Figure 4: Effect of Solanum nigrum polysaccharide on protein expression of caspase-3 and Bcl-2 in tumor tissue. Fifty mice bearing with H22 cells were randomly divided into five groups: model group, cyclophosphamide group (30 mg/kg), and Solanum nigrum polysaccharide groups with low, medium, and high doses of Solanum nigrum polysaccharide (30, 60, and 120 mg/kg). (a) Representative photograph of expression of caspase-3 and Bcl-2 in tumor tissue. (b) Statistical results of protein expression of caspase-3. (c) Statistical results of protein expression of Bcl-2. Data are expressed as the mean ± standard error mean n = 10; *P < 0.05 was considered statistically significant compared with model group

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 > Discussion Top


Malignant tumor threatens severely the health and life of human beings.[6] Most of the chemotherapy drugs lack of selectivity and have severe adverse reactions because they kill both tumor cells and the normal body cells simultaneously, consequently leading to the dysfunction of immune systems. However, TCM has the advantages of satisfied clinical effects of both preventing and treating tumors, low toxic and side effects, low resistance, and improvement of the life quality of patients. Therefore, finding the active ingredients or effective monomer compounds from the TCM can be regarded as a kind of new drug development pathway. The modern pharmacological studies confirmed that S. nigrum inhibits the growth of tumor cells by inducing the apoptosis of tumor cells and by enhancing the immune system.[8],[9],[10] Regarding the antitumor components in Solanum nigrum, S. nigrum alkaloid was confirmed to be responsible. However, S. nigrum alkaloid has reproductive toxicity, which limited the use for tumor treatment. Whether SNP, another active ingredient in S. nigrum, has an antitumor effect remains unclear.

In the present study, we found that SNP dose dependently inhibited the tumor growth in H22 tumor-bearing mice. H and E staining for pathological observation showed that tumor cells grew vigorously and had abundant dense and less area of necrosis. However, in the SNP groups, the number of tumor cells in the tumor tissue decreased and the tumor cells arranged sparsely with nuclear condensation or fragmentation and increased necrotic and apoptotic area.

The index of thymus and spleen is used to evaluate the inhibitory effect of drugs on immune function.[11],[12] This study showed that CTX exhibited the higher tumor inhibition rate while greatly damaged immune organ including spleen and thymus. However, SNP with low and high doses increased the index of the thymus. The results indicated that SNP exhibited both antitumor effect and immune function protection, which is consistent with the report.[13]

The imbalance between cell proliferation and apoptosis is an important mechanism of the occurrence and development of a malignant tumor, therefore inducing apoptosis is regarded as a new approach for antitumor drug research and development.[14] Cell apoptosis is closely regulated by many genes including oncogenes, proto-oncogene, and antioncogene.[15] The cysteine protease (caspase) family is a group of homologous proteinase system which directly leads to the disintegration of the apoptotic cell. Caspase-3, one of the important members in the family, is the key regulator of mitochondrial apoptosis, and the high expression of caspase-3 is closely related to the severe apoptosis.[3] Bcl-2 is localized mainly in the cell plasma membrane such as mitochondrial membrane, nuclear peripheral membrane, and endoplasmic reticulum.[16],[17] Bcl-2 is also the key regulator in mitochondrial apoptosis and is closely associated with mammalian cell apoptosis.[18] The overexpression of bcl-2 inhibits the apoptosis and delays the cell death.[18] Bcl-2 enhances the mitochondrial membrane potential by inhibiting the release of mitochondrial calcium ion and inactivating the endonuclease.[19] The present results showed that SNP increased the protein expression of caspase-3, while decreased the expression of bcl-2 in tumor tissue, which might contribute to the tumor inhibition by SNP.

In summary, the present study showed that SNP inhibited the growth of tumor in H22-bearing mice and protected the immune organ. The mechanism underlying the inhibition of tumor might be related to the upregulation of caspase-3 and downregulation of bcl-2, subsequently leading to the cell apoptosis.

Financial support and sponsorship

This study was supported by Scientific Research Foundation of Department of Education in Zhejiang Province (Y201431468) and Program of Jiaxing Municipal Sci & Tech Bureau (2016AY23096).

Conflicts of interest

There are no conflicts of interest.



 
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