Effects of low‑dose bufalin combined with hydroxycamptothecin on human castration‑resistant prostate cancer xenografts in nude mice
- Authors:
- Published online on: July 15, 2021 https://doi.org/10.3892/etm.2021.10447
- Article Number: 1015
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Copyright: © Gu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Prostate cancer is a high-risk malignant tumor of the urinary tract, typically diagnosed in middle-aged and elderly men (1). The worldwide incidence rate of prostate cancer accounted for 15% of the total number of malignant tumors in males in 2012(2), displaying a significant upward trend each year (3). Prostate cancer is the third largest cause of cancer-associated death in males in the United States (4) and the second largest cause worldwide (2). At present, the treatment of prostate cancer includes surgery, radiotherapy, chemotherapy and endocrine therapy (5). However, for advanced prostate cancer, surgery is ineffective and other treatment options are not satisfactory, with the majority being associated with severe adverse reactions (5). The main therapeutic strategy for prostate cancer is endocrine therapy, however, after a median of 18-20 months, the majority of patients will eventually develop androgen resistance, with a median survival of 12 months (6,7). Therefore, the treatment of castration-resistant prostate cancer (CRPC) has become a focus in urology research and the identification of novel treatment options is required (8).
Bufalin is a steroidal terpene compound, and is one of the active ingredients in traditional Chinese medicine (9). Bufalin has been reported to be useful in the treatment of liver and pancreatic cancers, as well as other tumors (9-12). Previous studies have reported that bufalin can significantly inhibit the proliferation of PC3 CRPC cells in vitro (13,14). However, the therapeutic dose is close to the toxic dose, which limits its clinical application (15,16). Bufalin is a DNA topoisomerase (TOP)II inhibitor (17), and its mechanism of action is related to the inhibition of tumor proliferation, metastasis and angiogenesis, as well as the reversal of tumor resistance (18-21). Cortés and Piñero (22) reported that DNA TOPI inhibition in ovarian cells by the TOPI inhibitor irinotecan resulted in increased levels of TOPII mRNA and protein expression, allowing DNA metabolism to continue (23,24). This phenomenon is called the side-channel sensitivity of TOP, and is required to maintain the normal physiological state of the cells (25,26). In oral cancer research, Ding et al (27) combined the TOPI inhibitor irinotecan and the TOPII inhibitor doxorubicin to improve treatment efficacy.
Previous studies have reported that the DNA TOPI inhibitor hydroxycamptothecin and the TOPII inhibitor bufalin can inhibit the growth of the CRPC cell line DU 145 in vitro (28,29). These studies also suggested that simultaneous administration of the two inhibitors was not as effective as individual drug administration, due to antagonism, but sequential administration significantly improved the results obtained (28,29).
The present study investigated whether bufalin in combination with hydroxycamptotecin would exhibit the same effect in vivo, as has been reported in previous in vitro studies. Furthermore, the present study aimed to identify a dose of low-toxicity bufalin combined with hydroxycamptothecin for further clinical applications.
Materials and methods
Cell lines and cell culture
The human prostate cancer cell line DU 145, purchased from Thermo Fisher Scientific, Inc., was cultured in RPMI 1640 complete medium (Sigma-Aldrich; Merck KGaA) containing 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA) and 1% penicillin and streptomycin solution, at 37˚C with 5% CO2 and fully saturated humidity. Cells were subcultured each for 2-3 days.
Establishment of a CRPC xenograft model in nude mice and treatment administration
A total of 41 male BALB/c nude mice (age, 22-25 g; 6-8 weeks), obtained from the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine were housed under pathogen-free condition at 22±2˚C with 40-60% humidity, 12-h light/dark cycles and free access to food and water. Cell suspensions of DU 145 cells in the logarithmic growth phase were prepared at a concentration of 1x107 cells/ml, using physiological saline. To induce tumor formation, five mice were subcutaneously injected in the abdomen with 0.2 ml cell suspension. At three weeks post-inoculation, when the diameters of the primary implanted tumors had grown to 1 cm3, tumors were removed and abdominally implanted into the other 36 mice. After 8 days, drugs were administered once every other day for 30 days. For drug administration, the 36 tumor-bearing mice were randomly divided into six groups, with six mice in each group. The groups were as follows: Normal saline negative control group (SN), hydroxycamptothecin (2 mg/kg; BioCrick) single drug positive control group (H), bufalin (1 mg/kg, Sigma-Aldrich; Merck KGaA) single drug positive control group (B) and hydroxycamptothecin (2 mg/kg) sequentially combined with 0.4 mg/kg bufalin treatment group (H4B), 0.6 mg/kg bufalin treatment group (H6B) or 0.8 mg/kg bufalin treatment group (H8B). No adverse reactions were reported in the mice in the combined treatment groups. The sequential co-administration method involved administration of the corresponding dose of bufalin 8 h after the administration of hydroxycamptothecin, as previously described (30). The present study was approved by the Institutional Ethics Committee of Shanghai University of Traditional Chinese Medicine.
Morphological and histological observation of tumors
A total of one day post-treatment, the six mice in each group were sacrificed and the tumors were completely removed. The tumors from each group were compared by morphological analysis. The long diameter (a) and short diameter (b) of the tumor were measured. Tumor parameters were calculated using the following formulae: Tumor volume (V; mm3)=(ab2)/2; tumor-inhibition rate (%)=[1-(V administration group/V negative control group)] x100; and tumor mass was determined using an electronic balance. A section of the tumor mass was removed, fixed in 10% formalin (pH 7.4) at room temperature for 24 h and embedded in paraffin. The paraffin-embedded samples were sectioned (4 µm). For pathological analysis, sections were stained at room temperature using haemotoxylin for 3 min and eosin for 30 sec (HE). Subsequently, tissue sections were examined using the Leica DM6 B light microscope (Leica Microsystems Inc.; magnification, x4, x10 and x40).
TUNEL detection
The TUNEL Assay kit-FITC (cat. no. ab66108; Abcam) was used to detect apoptosis in paraffin-embedded sections, according to the manufacturer's protocol. The staining of tumor cells in each group was observed under a fluorescence microscope (Leica DMLB; Leica Microsystems Inc.; magnification, x400) and necrotic areas were avoided. TUNEL-positive cells were observed in five randomly-selected high-power fields. The integrated optical density (IOD) values of the images were analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.) to assess the extent of apoptosis in tumor cells.
Detection of proliferating cell nuclear antigen (PCNA) protein expression by immunohistochemistry
Immunohistochemical staining was performed to detect the expression of PCNA in the xenograft tumors. Paraffin sections were rehydrated. The sections were subsequently treated as follows: Microwave antigen retrieval (700 W for 8 min; twice in 10 mM sodium citrate; pH 6.0) was followed by incubation with 3% hydrogen peroxide to block endogenous peroxidase and 10% goat serum (cat. no. 5560-0007; Seracare Life Sciences, Inc.) at 4˚C for 30 min to block nonspecific binding. PCNA was detected with rabbit anti-PCNA (1:100; cat. no. ab18197; Abcam) overnight at 4˚C and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:400; cat. no. ab205718; Abcam) for 1 h at room temperature. After that, 3,3-diaminobenzidine tetrahydrochloride (DAB; cat. no. 30015; Biotium, Inc.) was used for the chromomeric reaction, and hematoxylin was used to stain the nucleus for 5 min at room temperature. The staining of the tumor nuclei in each group was observed using a Leica DFC300 FX light microscope (Leica Microsystems Inc.; IL; magnification, x400). PCNA-positive cells, displaying brown-yellow granules in the nuclei, were observed in five randomly-selected high-power fields. The integrated optical density (IOD) values of the images were analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.). For the negative control, the primary antibody was replaced by normal rabbit IgG.
Western blotting
A homogenizer was used to prepare lysates from xenograft tumor tissues. The xenograft tumor tissues were lysed in radioimmunoprecipitation assay lysis buffer (Rockland Immunochemicals, Inc.) according to the standard protocol. Total protein was quantified using a bicinchoninic acid assay. Total protein lysate (50 µg) was loaded into each lane and separated using 12% SDS-PAGE gels. The separated proteins were then transferred onto PVDF membranes (Merck KGaA) and 3% BSA (cat. no. PRO-22; ProSpec-Tany TechnoGene, Ltd.) in TBS-Tween-20 (TBST) was added to the membranes for 1 h at room temperature. The membranes were washed three times with TBST and were subsequently incubated with diluted primary antibodies overnight. Primary antibodies including Bax (1:1,000; cat. no. 2772), Bcl-XL (1:1,000; cat. no. 2764), p53 (1:1,000; cat. no. 2527), PDCD4 (1:1,000; cat. no. 9535), GSK-3β (1:1,000; cat. no. 12456), p-AKT (1:2,000; cat. no. 4060), AKT (1:1,000; cat. no. 4685) and GAPDH (1:1,000; cat. no. 5174) were added and incubated overnight at 4˚C. Following the primary incubation, membranes were washed with TBST three times (x5 min) and then incubated with the horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:1,000; cat. no. 4030-05; SouthernBiotech) for 1 h at room temperature. After washing the membranes three times with TBST, Immunoblot detection and visualization were performed using enhanced chemiluminescence western blotting detection reagents (SuperSignal™ West Pico PLUS Chemiluminescent Substrate; cat. no. 34577; Thermo Fisher Scientific, Inc.). Immunoblotting was performed with target antibodies and protein bands were scanned and quantified using a ChemiDoc image analysis system (Bio-Rad Laboratories, Inc.). ImageJ software (version 1.46; Natioanl Institutes of Health) was used for densitometry analysis. All Primary antibodies were purchased from the Cell Signaling Technology, Inc.
Data analysis and statistical processing
Statistical analysis was performed using SPSS software (version 21.0; IBM Corp.). Data are presented as the mean ± standard deviation. All experiments were performed in triplicate. Data were analyzed by one-way ANOVA followed by Bonferroni post-hoc test. The Wilcoxon rank sum test was used to compare non-normally distributed data sets in non-parametric tests. The Mann-Whitney U method was used to test the significant differences between groups. P<0.05 was considered to indicate a statistically significant difference.
Results
Comparison of tumor size in different groups
After successful establishment of CRPC xenografts in mice, the mice were weighed and no statistically significant difference was observed between treatment groups (Table I; P>0.05). The mice in the treatment groups displayed no abnormal changes in body weight or behavior. The mice were sacrificed one day post-treatment and comparisons of tumor size of the xenograft tumors isolated from different groups were performed (Fig. 1A).
 | Table IComparison of the weight of nude mice, as well as the volume, weight and inhibition rate of the xenograft tumors. |
The volume and weight of the xenograft tumors were measured. All drug treatments significantly reduced the tumor volume compared with the SN group (P<0.05; Fig. 1B). Among the different drug treatment groups, the H6B and H8B groups were more effective at inhibiting increases in the tumor volume compared with the single drug administration groups (H or B) and the H4B group (P<0.05; Table I; Fig. 1B). However, the H6B group displayed the lowest tumor volume out of all of the groups (Table I; Fig. 1B). Similarly, in terms of tumor weight, the H6B and H8B groups were more effective at inhibiting tumor growth compared with all other groups (P<0.05; Table I; Fig. 1C). The tumor weight of the H4B and H groups was significantly reduced compared with the SN group (P<0.05; Table I; Fig. 1C). However, the B group did not display a significant difference in tumor weight compared with the SN group (P>0.05; Table I; Fig. 1C). Additionally, although the H6B group appeared to limit tumor weight to a further extent than the H8B group, the difference was not significant (P>0.05; Table I; Fig. 1C).
The tumor-inhibition rate of each group was calculated in each treatment group. The tumor-inhibition rate of the H6B and H8B groups was >80%, which was significantly higher than that of the single drug administration groups (P<0.05; Fig. 1D). The tumor-inhibition rate in the H6B group reached 92.99±3.96%, but there was no statistical difference between the H6B and H8B groups (P>0.05; Table I; Fig. 1D). The tumor-inhibition rate in the H4B group was not significantly different (P>0.05) from that in the H group, but was significantly (P<0.05) improved compared with the B group (Fig. 1D).
Pathological observation by HE staining
HE staining was performed on the xenograft tumors and suggested that the normal tissue structure was disorganized in each group, as indicated by disorderly cell distribution and different cell sizes. The nuclei morphology was irregular and several nuclei were much larger in the treatment groups compared with the SN group. In addition, the nucleus to cytoplasmic ratio was increased in the treatment groups compared with the SN group. In the drug-administered groups, obvious cavities were identified in the central part of the tumor cells and the cell size was different compared with the SN group. Furthermore, the drug-administered groups displayed no structural eosinophilic red staining or nuclear debris. The number of tumor cells in the cavity of drug-administered groups was reduced compared with the SN group, and it was impossible to distinguish apoptosis and necrosis. In the H, B and H4B groups, small changes to the structure and an interstitial connection in the cavity were observed, while in the H6B and H8B groups, severe cavity changes were observed and the original tissue structures could not be seen under the microscope. Therefore, it was speculated that the tumor suppressing effect of the two drugs was stronger in the combination therapy groups compared with the monotherapy groups (Fig. 2).
TUNEL assay for apoptosis
The TUNEL kit was used to detect apoptosis in the xenograft tumors of each group. The drug-administered groups displayed significantly higher levels of apoptosis compared with the SN group (P<0.05; Fig. 3A and B). Among the drug-administered groups, the H6B group displayed the highest level of apoptosis. The H8B group did not induce apoptosis to the same extent as the H6B group, but still displayed significantly (P<0.05) higher levels of apoptosis compared with the single drug administration groups (H and B) and the H4B group. No statistically significant (P>0.05) differences were found between the H4B group and the single drug administration groups (Fig. 3A and B).
Differential expression of PCNA protein
The PCNA protein is ubiquitously expressed in the nucleus, and its nuclear content is consistent with the synthesis of DNA, making PCNA an indicator of cell proliferation (31). The expression of PCNA in prostate cancer xenograft tumors was analyzed by immunohistochemistry. PCNA-positive cells were stained brown or yellow, indicating that the cells were proliferating and dividing (Fig. 4A).
The xenograft tumors of the drug-administered groups displayed significantly reduced PCNA levels compared with the SN group (P<0.05; Fig. 4A and B). The H6B and H8B groups displayed the lowest levels of PCNA expression compared with all the other treatment groups (P<0.05; Fig. 4A and B). There was no significant difference (P>0.05) between the PCNA expression in xenograft tumors from the H4B group and the single drug administration groups (H and B). The H6B group exhibited the most significant effect on PCNA expression, and there was a statistical difference compared with the single drug administration groups (P<0.05; Fig. 4B).
Expression of apoptosis-related proteins Bax, p53, programmed cell death 4 (PDCD4) and glycogen synthase kinase (GSK)-3β
To further explore the mechanisms of drug inhibition on tumor growth, the expression of cytoplasmic proteins in xenograft tumors was determined by western blotting. All drug treatments increased the protein expression levels of the tumor suppressor genes p53 and PDCD4, the mitochondrial apoptosis-related protein Bax and the PI3K/AKT/GSK-3β apoptosis signaling pathway-related protein GSK-3β, compared with the SN group (Fig. 5). Furthermore, all drug treatments decreased the protein expression levels of the mitochondrial apoptosis-related protein Bcl-XL and the PI3K/AKT/GSK-3β apoptosis signaling pathway-related protein p-AKT, compared with the SN group (Fig. 5). Additionally, the H4B, H6B and H8B groups significantly increased the protein expression levels of Bax, p53, PDCD4 and GSK-3β, and decreased the protein expression levels of Bcl-XL and p-AKT compared with the single drug administration groups (H and B; P<0.05; Fig. 5). Among the combination treatment groups, the changes were the most prominent in the H6B group, followed by the H8B and H4B treatment groups, respectively, although there was no significant difference among the three groups.
Discussion
The use of bufalin for the inhibition of tumor cell growth has been researched extensively in precious years (32-37). Low-dose bufalin displays an inhibitory effect on the growth of prostate cancer DU 145 cells in a time and dose-dependent manner (38). Administration of a combination of a specific dose of hydroxycamptothecin with a single agent was found to be effective against cancer cells (28). Similar synergy was also reported for the combination of hydroxycamptothecin and etoposide in human colon carcinoma HT-29 cells (39).
The present study suggested that in a nude mouse CRPC xenograft model, low-dose bufalin inhibited increases in tumor volume and weight, but bufalin (0.6 and 0.8 mg/kg) combined with hydroxycamptothecin had an improved effect on tumor volume, weight and inhibition rate compared with the administration of either drug alone. Histopathological analysis of sections of the xenograft tumors indicated increased cell death with the combined administration of bufalin and hydroxycamptothecin compared with the other groups. The TUNEL assay suggested that the H6B and H8B groups promoted higher levels of tumor cell apoptosis compared with the single drug administration groups. Immunohistochemical staining indicated that the H6B and H8B groups were more effective at inhibiting the proliferation of prostate cancer cells than all other treatment groups. The proapoptotic and growth-inhibiting effects of bufalin, hydroxycamptothecin or their combination may be related to the mitochondrial, p53-related and PI3K/AKT/GSK-3β apoptotic signaling pathways.
In recent years, bufalin has been reported to exhibit proapoptotic effects in a number of tumors (40-44,41), but the effective dose (≥1.5 mg/kg) utilized in previous studies is close to the toxic dose. According to the Dictionary of Traditional Chinese Medicine, the median lethal dose (LD50) of bufalin in nude mice is 2.2 mg/kg (45), and a number of previous studies have reported that 1.5 mg/kg bufalin significantly promoted the apoptosis of transplanted tumor cells and exhibited antitumor effects in nude mice (37,46,16,37). In the present study, the dose of bufalin used in the combination treatment groups (0.4, 0.6 and 0.8 mg/kg) was much lower than the LD50 value. There was no significant difference in the body weight of mice in the treatment groups compared with the SN group. The effects of the three combined treatment groups were no less than those of the bufalin (1.0 mg/kg) alone positive control group. Therefore, it can be suggested that the use of low-dose bufalin combined with hydroxycamptothecin may have a significant therapeutic effect, and may not be associated with toxicity, providing rationale for the clinical use of low-dose bufalin. However, the administration of a bufalin and hydroxycamptothecin combination would need to follow a specific protocol. The present study further suggested that the simultaneous administration of bufalin and hydroxycamptothecin was effective in the treatment of CRPC. The use of bufalin and hydroxycamptothecin simultaneously leads to drug antagonism, but sequential administration may lead to a synergistic effect (47). Therefore, administration of hydroxycamptothecin for a certain period of time prior to the administration of bufalin may be more effective (47). The present study suggested that the sequential administration of bufalin (0.6 and 0.8 mg/kg) 8 h after the administration of hydroxycamptothecin (2 mg/kg) was more beneficial. However, the role of hydroxycamptothecin in CRPC requires further investigation.
Bcl-XL and Bax belong to the Bcl-2 protein family (48). By controlling the permeability of the mitochondrial inner membrane structure, Bcl-XL and Bax affect proapoptotic factors in the cytoplasm, including cytochrome C, and transmit apoptotic signals to regulate cell death (49). Bax is an important component of mitochondrial membrane ion channels (50). After receiving the apoptotic signal, Bax expression is increased, proapoptotic factors in the mitochondria, such as cytochrome C, enter the cytoplasm and the caspase protein family is activated to induce apoptosis (51-53). Bcl-XL is primarily located in the cytoplasm and can be translocated to the mitochondrial outer membrane to bind Bax and form Bcl-XL/Bax heterodimers, under the action of apoptotic signals (20,21). Subsequently, the Bcl-XL/Bax heterodimers maintain the integrity of the mitochondrial outer membrane and interfere with apoptosis induction (54,55). The sequential administration of bufalin 8 h after the administration of hydroxycamptothecin enhanced the expression of Bax and inhibited the expression of Bcl-XL, potentially promoting apoptosis. The present study suggested that the combination of hydroxycamptothecin and bufalin, at the dose of 0.6 mg/kg, was the most beneficial treatment option.
Both p53 and PDCD4 are tumor suppressor genes, which play roles in cell apoptosis and DNA damage repair (56,57). Under physiological conditions, p53 levels are low in the cell (58). When DNA damage occurs in cells, p53 accumulates in the cells and promotes the apoptosis of abnormal cells via the p53/Bax apoptosis regulatory signaling pathway to prevent excessive proliferation of abnormal cells (59,60). The present study suggested that the apoptotic effect of bufalin on tumor cells is related to the activation of p53 and an increase in PDCD4 expression, which could potentially prevent the excessive proliferation of prostate cancer cells.
The PI3K/AKT signaling pathway is important for cell membrane receptor signaling (61). AKT regulates the proliferation of downstream proteins, including caspase 9, Bad, NF-κB and GSK-23, by phosphorylation, thereby regulating cell proliferation, differentiation, apoptosis and migration (62). GSK-3β can inhibit the expression of transcription factors, including β-catenin, Nrf2 and NFAT and activate the caspase pathway to induce apoptosis (63). The combination of bufalin and hydroxycamptothecin promoted the expression of GSK-3β and inhibited the expression of p-AKT, potentially inhibiting the growth of tumor cells. Furthermore, the combination of hydroxycamptothecin and bufalin at a dose of 0.6 mg/kg was the most effective at promoting GSK-3β expression and inhibiting p-AKT expression.
To conclude, sequential administration of bufalin and hydroxycamptothecin inhibited the growth of CRPC xenograft tumors. The dosage used for co-administration influenced the degree of drug inhibition. The administration of hydroxycamptothecin (2 mg/kg) followed by the administration of bufalin (0.6 mg/kg) 8 h later was the most effective treatment method assessed in the present study. The proapoptotic effect of bufalin and hydroxycamptothecin may occur via signaling pathways associated with mitochondrial apoptosis, PI3K/AKT/GSK-3β apoptotic signaling and p53-dependent apoptosis regulation.
Acknowledgements
Not applicable.
Funding
Funding: The present study was funded by the Shanghai Putuo Hospital, Shanghai University of Traditional Chinese Medicine (grant no. 2016233A).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
QZ performed the experimental work and collected and interpreted the data. RG designed the study, performed the analysis of the data, interpreted the data and drafted the manuscript. Both authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Institutional Ethics Committee of Shanghai University of Traditional Chinese Medicine.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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