Improved antitumor activity of cisplatin combined with Ganoderma lucidum polysaccharides in U14 cervical carcinoma‐bearing mice

Abstract

Research on anticervical cancer is urgently required to enhance clinical outcomes. As a main anticancer drug for cervical carcinoma, cisplatin (CIS) has been used for a lot of years in clinical therapy. However, serious adverse effects including nephrotoxicity and neurotoxicity limit its long‐term treatment. Our main goal of this study is to investigate the improvement of Ganoderma lucidum polysaccharides (GPS) on CIS‐induced antitumor effect of in U14 cervical carcinoma‐bearing mice. The results showed that GPS + CIS could not only inhibit the growth of the tumor but also improve the spleen and thymus indexes. Moreover, little toxicological effects were observed on hepatic function and renal function in GPS + CIS treated mice bearing U14 tumor cells. Further analysis of the tumor inhibition mechanism indicated that the number of apoptotic tumor cells increased significantly, the expression of Bax increased and the expression of Bcl‐2 decreased dramatically in cervical cancer sections after oral administration of GPS + CIS for 14 days. This GPS/CIS combined therapy represents intriguing therapeutic strategy for U14 cervical carcinoma providing not only superior efficacy but also a higher safety level.

1. INTRODUCTION

Cervical cancer is a prevalent malignant tumor in female, which has more than 500 000 new cases diagnosed each year all around the world, seriously affecting female's health and life.1 At present, radiotherapy, surgery, and chemotherapy are the main ways in the treatment of this cancer. However, the surgical treatment is only applicative for the young patients and these patients in the early stage.2 Moreover, chemotherapy and radiotherapy often cause lots of adverse effects, including immunological and organ toxicity.3 Cisplatin (CIS) is a DNA‐damaging antitumor drug and commonly used in chemotherapy owes to prominent effective in treating neck, cervical, ovarian, and testicular cancers.4 CIS establishes cross links between DNA with purine bases and interferes with DNA restore mechanisms, causing DNA injury, and ultimately inducing apoptosis in carcinoma cells.5 However, the CIS also induced undesirable adverse effects including kidneys damage, liver function disorder, and allergic reactions, its efficacy is restricted. Hence, novel therapeutic strategies are urgently required to alleviate those side effects and enhance CIS antitumor effect which would be significant and will decrease the adverse effects of CIS treatment.

Presently, there are various researches have reported that combining chemotherapy with natural products might improve the antitumor effect, decrease the side effects of chemotherapy, and improve quality of life.6, 7 Natural polysaccharides are the main bioactive ingredient in natural medicine.8 Ganoderma lucidum is an edible mushroom and has long been served as a Traditional Chinese Medicine with medicinal properties. Polysaccharides are known the main composition of G. lucidum and possess various pharmacological activities, such as immunomodulatory, antitumor, liver protection, and anti‐inflammatory.9 However, it remains to be investigated whether the combination of G. lucidum polysaccharides and CIS can be applied to cervical cancer treatment.

The previous study has reported that the G. lucidum polysaccharides possess the activities of immunomodulatory and antioxidant in cervical carcinoma rat.10 In the present research, we hypothesized that G. lucidum polysaccharides could assuage CIS treatment induced immunosuppression and organ damage, and to improve the antitumor activity of CIS. Hence, the purpose of the present study to demonstrate whether the combined use of G. lucidum polysaccharides (GPS) and CIS could be effective in cervical carcinoma treatment, therefore we investigated the effects of G. lucidum polysaccharides plus CIS combination in U14 cervical carcinoma‐bearing mice.

2. MATERIAL AND METHODS

2.1. Chemicals and reagents

G. lucidum was purchased from Fujian Xianzhilou Biotechnology Co., Ltd. (Fujian Province, China) and was identified as artificial cultivar. Cisplatin was purchased from Qilu Pharmaceutical Co., Ltd. (Shandong Province, China); eosin, hematoxylin, and rabbit polyclonal antibody to Bcl‐2 (N‐19), Bax (P‐19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Calf serum and RPMI‐1640 were purchased from Invitrogen Inc. (Carlsbad). All other reagents were purchased from Sigma‐Aldrich Inc. (Louis).

2.2. Preparation of GPS

G. lucidum polysaccharide (GPS) was prepared from the fruiting body of G. lucidum by traditional hot water extraction according to method with minor modification.11 Briefly, the extraction condition was: an extraction temperature of 95°C, water/solid of 15:1, and extraction time of 3 hours. The extract was centrifuged to obtain the supernatant and precipitated with 85% ethanol at 2°C for 12 hours after concentration under vacuum. GPS was obtained by lyophilized and deproteinated by Sevag method. The content of polysaccharides was 90.13% and measured by phenol‐sulfuric acid method.

2.3. Cell line and animals

Mouse cervical cancer cell line U14 obtained from Institute of Material Medical (Chinese Academy of Medical Sciences, China). Female Kunming specific pathogen free mice (18‐22 g weight and 6‐8 weeks old) were purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences. The mice were maintained in a constant temperature (20 ± 2°C), and fed with a basic diet and water in plastic cages. Animal experimentation was carried out in according to the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the local animal Ethics Committee.

2.4. Animal model and treatment

All mice were fasted for 12 hours before the establishment of cervical carcinoma model, and then U14 carcinoma cells at logarithmic phase were propagated in abdominal cavity of Kunming mice for 7 days. The ascites were taken out from the mice and diluted with sterile normal saline to 2 × 10⁷ cells/mL. Then, the subcutaneous injection of 0.2 mL carcinoma cell suspension was conducted into the right axilla to establish U14 carcinoma model. After 24 hours of inoculation, animals were randomly into four groups (10 mice per group) as follows: model control group (MC), injection of sterile saline was given around the tumors; cisplatin group (CIS), injection of 5 mg/kg cisplatin was given around the tumor; G. lucidum polysaccharide group (GPS), injection of 30 mg/kg GPS was given around the tumors while the combined injection of GPS and cisplatin was given for CIS + GPS group with the same doses as CIS group and GPS group. All the mice were treated one time daily for 2 weeks. After 24 hours of the last administration, all the animals were executed. Then, the samples of thymus, spleen and tumor tissues were immediately collected and weighted. The tumor inhibition ratio was counted according to the following formula: [(the mean tumor weight of model control group − the mean tumor weight of treated group)/the mean tumor weight of model control group] × 100%. The organ indexes for the spleen and thymus were calculated using the following formula: Organ index = thymus or spleen gland weight/body weight.

2.5. Observation of tumor volume and survival time

The width and length (mm) of tumor were measured with calipers every 3 days, and the tumor volume was calculated by the following formula: tumor volume (mm³) = length × width² × 0.5, where width is the perpendicular short diameter and length is the longest diameter. Besides, another four groups of mice were treated as the above protocol for survival time study. All mice were observed for 28 days after the U14 cervical carcinoma challenge.

2.6. Analysis of liver and renal function

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), serum creatinine (CRE), and serum urea nitrogen (BUN) were determined by commercial assay kits on the basis of manufacturer's specification (Nanjing Jiancheng Bioengineering Institute).

2.7. Apoptosis cells measurement by terminal deoxynucleotide‐transferase‐mediated digoxigenin triphosphate‐biotin nick end labeling assay (TUNEL)

All tumor sections of mice were dewaxed and processed. TUNEL staining assay was performed using an In Situ Cell Death Detection Kit on the basis of manufacturer's instructions. Briefly, the positive cells whose nucleus appeared a distinct brown were considered apoptotic cells. The number of positive cells was counted semi‐quantitatively by averaging the number of apoptotic cells/field under a 400× magnification. Five fields were selected for per a sample, the apoptosis cells index was calculated as follows: apoptosis cells index = the number of positive cells/the total number of cells × 100%.

2.8. Immunohistochemical analysis of the expression of Bax and Bcl‐2

The collected tumor tissue of mice was fixed in 10% formalin, and embedded in paraffin. Tumor tissue sections were stained use a standard immunohistochemical method (streptavidin peroxidase conjunction) and observed under a light microscope (Nikon, Japan). The positive cells whose nucleus appeared a distinct brown; negative cells whose nucleus turned a distinct blue. The numbers of positive cells were counted use a hemacytometer and the mean number was calculated.

2.9. Detection of T‐lymphocyte subpopulations and white blood cells (WBC) count in U14 tumor bearing mice

By the end of the experiment on day 15, peripheral blood of all mice was collected for examination immediately after the animals were killed. Thymocyte suspensions were produced by filtering samples through a mesh screen and incubated with RBC lysis buffer for 20 minutes, then centrifugation at 1500 rpm for 5 minutes after diluted with PBS. Lymphocytes were measured and incubated for 30 minutes at 37°C with PE‐CD4⁺ and FITC‐CD8⁺ antibodies in the dark. Cells were analyzed with CELLQuest Pro software (BD Biosciences, CA). The white blood cells (WBC) were measured using the blood cell counting apparatus (Mindray, China).

2.10. Statistical analysis

All experimental results were reported as the means ± SD and the experiment were replicated twice. The differences between groups were estimated by one‐way anova and using SPSS software (version 16.0); P < 0.05 was usually considered as statistically significant.

3. RESULTS

3.1. Treatment effects on tumor growth of U14‐bearing mice

In order to evaluate the antitumor effect of CIS and GPS alone or in combination. The mice were transplanted with U14 carcinoma cells and the antitumor effect was investigated (Table 1, Figure 2B,D). CIS alone treatment obviously inhibited tumor growth (59.97% tumor inhibition rates; P < 0.01 vs model group). Compare with the model group, GPS alone treatment also suppressed tumor growth (33.34% tumor inhibition rates; P < 0.01 vs model group). Compared with the CIS alone treatment, the tumor inhibition rates of the combination treatment exhibited trends of being higher than that in the CIS group (P < 0.05 vs model group). These results showed that the effect of GPS + CIS is better than CIS treatment alone in inhibiting U14 tumor growth, implying that effect of CIS in tumor suppression may be potentially improved by GPS.

Table 1.

Comparison of WBC count, spleen index, thymus index, tumor weight, and tumor inhibition after medication

Groups	MC	CIS	GPS	CIS + GPS
WBC count (109/L)	9.73 ± 0.37	4.62 ± 0.29**	4.85 ± 0.45**	3.13 ± 0.23***
Spleen index (mg/g)	4.83 ± 0.91	3.63 ± 1.05**	7.59 ± 0.95**	6.93 ± 0.89****
Thymus index (mg/g)	2.27 ± 0.39	1.06 ± 0.27**	4.10 ± 0.60**	3.34 ± 0.47****
Tumor weight (g)	3.58 ± 0.32	1.43 ± 0.22**	2.08 ± 0.23*	0.91 ± 0.16***
Inhibition rate (%)	0	59.97 ± 5.04**	33.34 ± 7.45**	66.93 ± 3.76***

The data were expressed as the mean ± SD (n = 10/group).

^* P < 0.05 (vs the model group).

^** P < 0.01 (vs the model group).

^*** P < 0.05 (vs the CIS group).

^**** P < 0.01 (vs the CIS group).

Figure 2.

Effects of CIS treatment with or without GPS on apoptotic cells in tumors of U14‐bearing mice (A). The tumors image are displayed in B. Representative photomicrograph of the TUNEL staining (magnification: 400×) (C). Effects of CIS association with or without GPS on tumor volume curve in U14‐bearing mice (D). The data are reported as the mean ± SD (n = 10/group). ^*** P < 0.001 (vs the model group)

3.2. Treatment effects on immune organs index of U14‐bearing mice

To investigate potential immunomodulatory effects of CIS treatment with or without GPS, the spleen and thymus indexes of treated mice were investigated. As displayed in Table 1, compared with the model group, the spleen and thymus index were obvious decrease in CIS group (P < 0.05), whereas there was an obvious increase in the thymus index and a notable decrease in the spleen in the GPS alone or GPS + CIS groups (P < 0.05). These results implied that immunosuppression could be caused by CIS treatment and that GPS may potentially prevent the immunosuppressive effect of CIS to certain a degree.

3.3. Treatment effects on the T cells subsets in peripheral blood

Immunomodulatory actions of CIS treatment with or without GPS were further investigated on the cellular level. As showed in Figure 1A,C, compared to model group, the proportions of CD4⁺ T cells in peripheral blood and the CD4⁺/CD8⁺ ratio were markedly decreased in the CIS group (P < 0.05), implying its cytotoxicity on tissue cells. However, after treated with GPS alone or combination treatment, the proportions of CD4⁺ T cells and the CD4⁺/CD8⁺ ratio were obviously increased while compared with model group (P < 0.05), which showed that their capacity of antitumor effects by improving T cells immune function.

Figure 1.

The percentages of CD4⁺ (A), CD8⁺ (B) lymphocytes in the peripheral blood and CD4⁺/CD8⁺ lymphocytes ratio (C) detected by flow cytometry. The survival curve of mice after U14 tumor challenge (D). The data are reported as the mean ± SD (n = 10/group). ^* P < 0.05 (vs the model group), ^** P < 0.01 (vs the model group)

3.4. Effect of combined therapy on survival in U14‐bearing mice

As showed in Figure 1D, the survival rate of all groups of mice was observed after U14 cervical carcinoma challenge. The survival rate of U14 cervical carcinoma‐bearing mice treatment with GPS alone or CIS alone was obviously higher compared with the mice in MC group (P < 0.05). Furthermore, the mice of combined therapy group had an obviously higher survival rate than the mice in GPS group or CIS group (P < 0.05).

3.5. Treatment effects on WBC count of mice

As displayed in Table 1, CIS alone treatment obviously decreased the WBC count (P < 0.01 vs model group). Compare with the model group, GPS alone treatment also decreased the WBC count (P < 0.01 vs model group). Compared with the CIS alone treatment, the WBC count of the combination treatment exhibited trends of being lower than that in the CIS group (P < 0.05 vs model group).

3.6. Treatment effects on apoptosis of tumor cells

Apoptosis of tumor tissue section was further measured by the TUNEL straining method. As displayed in Figure 2A,C, the number of apoptotic cells was obviously increased by the administration of GPS or CIS alone compared with the model group (P < 0.001). The maximum apoptosis of tumor cells was observed in the CIS and GPS combination treatment group was higher than that in the model group (P < 0.01). These results demonstrated that the apoptosis of tumor cells was induced by treatment of CIS + GPS.

3.7. Treatment effects on protein expression of Bax and Bcl‐2 in tumor tissues

In order to further investigate the mechanism of CIS + GPS mediated apoptosis in tumor tissue. The protein expression of Bax and Bcl‐2 was measured by immunohistochemical assay. As showed in Figure 3, the level of the proapoptotic protein Bax was obviously increased and the level of antiapoptotic protein Bcl‐2 was significantly decreased in the CIS or GPS treatment group when compared with the model group (P < 0.05; P < 0.01). Moreover, in the CIS + GPS group, the protein level of Bax was highest and the level of Bcl‐2 was the lowest of the four groups. These results implied that GPS + CIS induced apoptosis is mediated by modulating Bcl‐2 and Bax family protein in tumor tissues of U14‐bearing mice.

Figure 3.

Effects of CIS association with or without GPS on expression of Bcl‐2 (A, C) and Bax (B, D) in tumors of U14‐bearing mice (Immunohistochemical analysis, magnification: 400×). The data are reported as the mean ± SD (n = 10/group). ^* P < 0.05 (vs the model group), ^** P < 0.01 (vs the model group)

3.8. Toxicological effects of CIS + GPS treatment in U14‐bearing mice

The perfect chemotherapeutics not only possess the advantage of inhibiting the growth of tumor cells but also have the minimum toxicity to their tissue. Hence, the serum levels of hepatic function and renal function indicators, such as ALT, AST, BUN, CRE were determined to evaluate the toxicological effects of CIS + GPS treatment on U14‐bearing mice (Figure 4). CIS alone treatment obviously increased the serum levels of ALT, AST, BUN and CRE when compared with the model group (P < 0.01). More importantly, these indicators were all obviously downregulated by CIS + GPS combination treatment as compared to the CIS group (P < 0.01, P < 0.05). And they were not significantly different as compared with the model group (P > 0.05). Collectively, these results implied that the administration of CIS + GPS shows very minor toxicological effects on U14‐bearing mice.

Figure 4.

Effects of CIS treatment with or without GPS on the serum levels of ALT (A), AST (B), BUN (C), and CRE (D) of U14‐bearing mice. The data are reported as the mean ± SD (n = 10/group). ^*** P < 0.001 (vs the model group)

4. DISCUSSION

Conventional chemotherapy has the limit as single modalities that play a vital role in the entirely treatment of cancer. Hence, novel strategies of carcinoma treatment by combined therapies are regarded as more potential for improving the antitumor efficacy, which could result in lower organs toxicity and longer survival time. In recent researches, more natural products including plant polysaccharides have been indicated to inhibit the activity of the tumor. In addition, conventional chemotherapy combined with these active ingredients may possess higher antitumor activity.12 G. lucidum one of the most popular edible mushroom and has long been used as a Traditional Chinese Medicine. G. lucidum polysaccharides (GPS) have been showed by a recent study to exert antitumor activity and result in attracting considerable attention.9 Therefore, in the present study, we investigated the improvement effects of adjuvant treatment with GPS in U14 bearing mice. Our results indicated that the combination of CIS with GPS improves the antitumor effect of CIS and lower organs toxicity in U14‐bearing mice.

Previously study has shown that novel polysaccharides isolated from G. lucidum possess higher antitumor activity by combination with cyclophosphamide in sarcoma 180‐bearing mice13 and G. lucidum polysaccharides could improve chemotherapy‐related fatigue via regulation oxidative stress and reduction of nephrotoxicity, these results indicated the synergistic effects of GPS on antitumor effect with cisplatin.14 Similarly, in our present research, we observed that GPS treatment alone also suppressed the growth of U14 cervical carcinoma. Moreover, GPS and CIS combination administration indicated a trend of possessing a higher capacity for suppressing U14 cervical tumor growth than CIS administration alone, which implying that the antitumor effect of the CIS could be enhanced by GPS supplementation.

Apoptosis is a vital homeostatic mechanism for a host to equilibrate the cell death and cell division, and as a consequence it is employed by the host to sustain the proper number of cells in the tissue. Induction of cancer apoptosis is a common phenomenon by anticancer treatment and has been recognized as an effective strategy for the development of anticancer drug.15 The current research showed that GPS alone or combination with CIS suppressed the growth of tumor in association with apoptosis. Apoptosis is regulated by proapoptotic and antiapoptotic effectors, which involves a lot of proteins. Bcl‐2 family proteins, such as the antiapoptotic protein Bcl‐2 and the proapoptotic protein Bax act as vital control points in the mitochondrial‐mediated apoptotic pathway.16 These results of the current research indicated that combined therapy indicated a tendency of possessing a higher capacity in down‐regulating Bcl‐2 expression and up‐regulating Bax expression in the tumor tissues than CIS treatment alone. These interesting findings suggest that GPS could enhance CIS‐induced apoptosis may be in connection with the effects of GPS on the change of Bcl‐2 family proteins.

In general, chemotherapy‐induced immunosuppression results in obvious mortality and morbidity, which becomes a principal limitation for its clinical application and without effective remedies so far. G. lucidum polysaccharides have been proven exert diverse pharmacological activities, particularly immunomodulatory effect.17 In the present study, our data demonstrated that CIS administration can cause immunosuppression (lower spleen index and thymus index compared to model group). The spleen and thymus are major tumor‐related immune organs. Spleen, the largest secondary immune organ in the body, and it plays a vital role in sustaining immune homeostasis.18 Thymus is the major hematopoietic site for T cells differentiation and maturation of CD8⁺ and CD4⁺ cells, which are mainly participators in the adaptive immune system.19 In this research, we found that GPS could obviously protect the immune organs and assuage the immunosuppressive effect caused by the combination CIS chemotherapy. Previous studies also obtained a similar tendency of alterations in the spleen index and thymus index as in our research.20, 21 The CD8 and CD4 glycoproteins are two major subsets of T lymphocytes.22 CD8⁺ T cells play a critical role in human antitumor immunity.23 CD4⁺ T cells could activate and recruit innate immune cells including macrophages and natural killer cells in human antitumor immunity.24 In this work, CIS treatment obviously reduced the proportions of CD4⁺ T cells in peripheral blood and CD4⁺/CD8⁺ T cell ratio. Furthermore, GPS + CIS combination treatment considerably assuaged CIS effect in decreasing the proportions of CD4⁺ T cells and the ratio of CD4⁺/CD8⁺. Our results imply that immunosuppression can be due to CIS treatment, which can be obviously assuaged by GPS supplementary treatment in U14‐bearing mice. Previously, some reports also shown that the proportions of CD4⁺ T cells in peripheral blood and the ratio of CD4⁺/CD8⁺ could be increased by treatment with the polysaccharides from Polygonatum sibiricum and Litchi pulp, and this finding was consistent with our results.25, 26 Based on the results of CIS + GPS on WBC count of carcinoma‐bearing mice, it was indicated that CIS as immunosuppressive agent could reduce the WBC count of carcinoma‐bearing mice. Interestingly, GPS or CIS + GPS could also reduce the WBC count of carcinoma‐bearing mice compared to the MC group. Indeed, the WBC count is an indicator of immunosuppression, it is also another marker of nonspecific inflammation. The effect of GPS or CIS + GPS on suppressing WBC count is not relevant to immune organs protective effect possibly due to the anti‐inflammatory activity of GPS.27 The previous studies showed that tumor‐induced increased of WBC count, and anti‐inflammatory ingredients Brucea javanica Oil or the glycyrrhetinic acid modified curcumin‐loaded cationic liposomes could suppress WBC count. These findings were consistent with our results.28, 29

Cisplatin (CIS) is a synthetic antitumor drug commonly used in clinically chemotherapy for the treatment of several malignancies. However, nephrotoxicity and hepatotoxicity remain the serious drawback in high dose of CIS chemotherapy.30, 31 BUN and CRE are the principal waste products that are eliminated from the kidneys. The increase in serum levels of BUN and CRE is used as a marker of CIS‐induced nephrotoxicity.32 In addition, the obvious increase of the serum levels of AST and ALT is commonly used as an indicator for liver damage.33 In present research, the results showed that treatment CIS to U14‐bearing mice induced an obvious increase in the serum levels of AST, ALT, BUN, and CRE, indicating that acute liver and renal failure, which can be obviously ameliorated by GPS supplementary treatment. These findings are consistent with previous studies that G. lucidum fruiting bodies protects kidney and liver function.27, 34

In conclusion, the combined therapy of GPS and CIS indicates a better antitumor activity than GPS or CIS treatment alone in U14‐bearing mice. In addition, GPS and CIS combination treatment can increase spleen and thymus indices, and improve body immunity by the promotion of T lymphocytes subsets, the CD4⁺/CD8⁺ T‐cell ratio when compared to CIS treatment alone. Furthermore, the combination treatment can improve liver and renal function when compared to CIS treatment alone. This research implies that combination therapy of GPS with CIS can enhance the antitumor activity of CIS and can assuage the immunosuppression and organ toxicity caused by CIS treatment in U14‐bearing mice, which represent potentially useful medicinal functions in prevention of cervical cancer.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Zhu J, Xu J, Jiang L‐L, et al. Improved antitumor activity of cisplatin combined with Ganoderma lucidum polysaccharides in U14 cervical carcinoma‐bearing mice. Kaohsiung J Med Sci. 2019;35:222–229. 10.1002/kjm2.12020