Abstract
Background/Aim. The present study determined the efficacy of extracts of Astragalus membranaceus (AM) and Curcuma wenyujin (CW), a traditional Chinese medicine herbal mixture, at different tumor stages of an orthotopic nude mouse model of human ovarian cancer expressing red fluorescent protein. Materials and Methods: The tumor-bearing mice were treated with cisplatinum (CDDP), AM, CW, or a combination of AM and CW in each of three tumor stages, using the same regimen. Group 1 received saline as negative control. Group 2 received CDDP i.p. as positive control with a dose of 2 mg/kg, every three days. Group 3 received AM daily via oral gavage, at a dose of 9120 mg/kg. Group 4 received CW daily via oral gavage, at a dose of 4560 mg/kg. Groups 5, 6 and 7 received combinations of AM and CW daily via oral gavage at low (AM, 2280 mg/kg; CW, 1140 mg/kg), medium (AM, 4560 mg/kg; CW 2280 mg/kg), and high (AM, 9120 mg/kg; CW, 4560 mg/kg) doses. The expression of angiogenesis- and apoptosis-related genes in the tumors were analyzed by immunohistochemistry for matrix metalloproteinase 2 (MMP-2), vascular endothelial growth factor (VEGF) fibroblast growth factor 2 (FGF-2), B-cell lymphoma 2 (Bcl-2) and cyclooxygenase 2 (Cox-2), and by polymerase chain reaction for MMP-2, FGF-2 and Bcl-2. Results: CDDP, AM, and its combination with CW-induced significant growth inhibition of Stage I tumors. Strong efficacy of the combination of AM and CW at high dose was observed. Monotherapy with CDDP, AM, CW, and the combination treatments did not significantly inhibit Stage II and III tumors. The expression of MMP-2, VEGF, FGF-2, and Cox-2 was significantly reduced in Stage I tumors treated with AM, CW, and their combination, suggesting a possible role of these angiogenesis- and apoptosis-related genes in the observed efficacy of the agents tested. Conclusion: This study is the first report on the efficacy of anticancer agents at different stages of ovarian cancer in an orthotopic mouse model. As the tumor progressed, it became treatment-resistant, similar to the clinical situation, further demonstrating the utility of the model and the need for agents acrtive in advanced-stage ovarian cancer.
- Astragalus membranaceus
- Curcuma wenyujin
- ovarian cancer
- red fluorescent protein
- orthotopic
- nude mice
- tumor progression
- metastasis
In 1993, we reported the first orthotopic transplant model for human ovarian cancer. Histologically-intact patient specimens of ovarian cancer were transplanted by microsurgical techniques under the capsule of the nude-mouse ovary. The human tumors grew locally and gave rise to a patient-like metastatic pattern, including the parietal peritoneum, colon, omentum, and ascites (1, 2).
We subsequently developed a green fluorescent protein (GFP)-expressing orthotopic metastatic nude-mouse model of the human ovarian cancer cell line SKOV3-GFP, enabling noninvasive imaging of tumor progression and metastasis (3).
The present study investigated the efficacy of a traditional Chinese medicinal herbal mixture on the various stages of an orthotopic nude-mouse model of human ovarian cancer expressing red fluorescent protein (RFP), enabling noninvasive imaging of tumor progression (3-5).
Staging of orthotopic mouse model of human ovarian cancer. In vivo fluorescence imaging and histological examination were performed to stage the orthotopic human HO-8910-RFP ovarian tumor mouse model. Panels depict whole-body noninvasive fluorescence imaging, open imaging at autopsy and H'E staining of the orthotopic ovarian tumor in a representative mouse at each of three stages.
Efficacy of Astragalus membranaceus (AM) and Curcuma wenyujin (CW) on Stage I tumor growth in the orthotopic human HO-8910-RFP ovarian cancer mouse model. Tumor growth was monitored and quantified by real-time noninvasive whole-body fluorescence imaging. Tumor was dissected and final tumor weight was determined at autopsy. A: Sequential in vivo whole-body fluorescence imaging of tumor on day 0, 10 and 17 after treatment initiation in mice treated with normal saline (NS), cisplatinum (CDDP), AM, CW, or low- (L), medium- (M), or high-dose (H) AM+CW. B: Tumor growth curves for mice treated with NS, CDDP, AM, CW and their combination. C: Final tumor weights at the end of the study in the mice of control and treated groups.
The dried root of Astragalus membranaceus (Fischer) Bge. var. mongholicus (Bge.) Hsiao (AM) is a traditional Chinese medicine (TCM). AM is frequently prescribed in many formulations and has been reported to possess diverse biological activities, including cardioprotective (6, 7), immunomodulatory (8, 9), anti-inflammatory and anti-cancer (10, 11). AM had significant growth-inhibitory, pro-apoptotic and angiogenesis suppression on colon, breast, hepatocellular, gastric, prostate and cervical human cancer cells in vitro (12-18). In addition, AM inhibited growth of colon and breast cancer in vivo (12, 19).
A. Efficacy on metastasis of Stage I tumors.
B. Efficacy on metastasis of Stage II tumors.
C. Efficacy on metastasis of Stage III tumors.
Efficacy of Astragalus membranaceus (AM) and Curcuma wenyujin (CW) on Stage II tumor growth in the orthotopic human HO-8910-RFP ovarian cancer mouse model. Tumor growth was monitored and quantified by real-time noninvasive whole-body fluorescence imaging. Tumor was dissected and final tumor weight was determined at autopsy. A: Sequential in vivo whole-body fluorescence imaging of tumor on day 0, 8 and 14 in mice treated with normal saline (NS), cisplatinum (CDDP), AM, CW, or low- (L), medium- (M), or high-dose (H) AM+CW. B: Tumor growth curves for mice treated with NS, CDDP, AM, CW and their combination. C: Final tumor weights at the end of the study in the mice of control and treated groups.
Curcuma wenyujin Y.H. Chen et C. Ling (CW), a widely prescribed TCM in clinical cancer therapy, also has antimicrobial (20) antiinflammatory (21, 22), and antiproliferative acitivies (20, 23-26). CW significantly inhibited uterine-cervical, prostate, gastric and breast cancer cell proliferation in vitro, and inhibited tumor growth in mouse models (27-30).
We previously reported proliferation inhibition and apoptosis induction by CW in HO-8910 ovarian cancer cells in vitro (31). Previous in vivo studies on AM and CW were performed on subcutaneous xenograft tumor models, which have limited clinical relevance.
In the present report, tumor progression in an orthotopic mouse model of ovarian cancer was classified into three stages by in vivo fluorescence imaging and histology in order to minic the clinical situation. The efficacy of AM and CW on each stage of tumor progression was evaluated. Expression of angiogenesis- and apoptosis-related genes in the tumors treated with AM and CW was evaluated to determine the mechanism of action of these herbal agents.
Materials and Methods
Cell culture. The HO-8910 human ovarian cancer cell line expressing RFP (HO-8910-RFP) (AntiCancer, Inc., San Diego, CA, USA), was cultured in RPMI-1640 medium (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) at −37°C in an atmosphere of 5% CO2 and saturated humidity. The medium was supplemented with penicillin/streptomycin (Gibco Life Technologies).
Animal care. BALB/C female nude mice (n=168), aged 4-6 weeks, weighing 20-25 g, were purchased from the Shanghai Laboratory Animal Center (SLAC) (Shanghai, China). All mice were maintained in a HEPA-filtered environment at 24-25°C and humidity was maintained at 50-60%. All animals were fed with autoclaved laboratory rodent diet. Animal experiments were approved by the Animal Committee of Nanjing Origin Biosciences (Nanjing, China) (OR1305).
Orthotopic mouse model. An orthotopic mouse model of HO-8910-RFP human ovarian cancer cell line was used for the present study (1-3). HO-8910-RFP stock tumor was established by subcutaneously injecting HO-8910-RFP cells (5×106) into the flank of nude in mice. Ovarian tumors grown s.c. in nude mice were harvested in the exponential growth phase and resected under aseptic conditions. Strong RFP expression of the HO-8910-RFP tumor tissue was confirmed by fluorescence microscopy. Necrotic tissues were removed and viable tissues were cut with scissors and minced into 1 mm3 pieces. Animals were anesthetized by injection of a solution of 50 mg/ml ketamine, 38 mg/ml xylazine, and 1.2 mg/ml acepromazine maleate (0.02 ml). The surgical orthotopic implantation (SOI) method followed our published procedures (1-3, 32). An incision approximately 1 cm long was made in the right lower abdomen of the nude mouse below the kidney using sterile scissors. The ovary was exposed and the capsule of the ovary was carefully opened. One ovarian tumor fragment was sutured to the ovary with 8-0 surgical sutures. The abdomen was closed in layers with sterile 5-0 surgical sutures. All surgical procedures and animal manipulations were conducted under aseptic conditions in a HEPA-filtered laminar-flow hood under a surgical microscope (Model YZ20P5, Shanghai Precision Instruments, Shanghai, China).
Efficacy of Astragalus membranaceus (AM) and Curcuma wenyujin (CW) on Stage III tumor growth in the orthotopic human HO-8910-RFP ovarian cancer mouse model. Tumor growth was monitored and quantified by real-time whole-body fluorescence imaging. Tumor was dissected and final tumor weight was determined at autopsy. A: Sequential in vivo non-invasive whole-body fluorescence imaging of tumor on day 0, 11 and 15 after treatment initiation in mice treated with normal saline (NS), cisplatinum (CDDP), AM, CW, or low- (L), medium- (M), or high-dose (H) AM+CW. B: Tumor growth curves for mice treated with NS, CDDP, AM, CW and their combination. C: Final tumor weights at the end of the study in the mice of control and treated groups.
In vivo fluorescence imaging. A fluorescence stereo microscope (MZ650; Nanjing Optic Instrument Inc., Nanjing, China) equipped with D510 longpass and HQ600/50 bandpass emission filters (Chroma Technology, Brattleboro, VT, USA) and a cooled color charge-coupled device camera (QImaging, Surry, BC, Canada) were used. Selective excitation of RFP was produced through an illuminator equipped with HQ470/40 and HQ540/40 excitation bandpass filters (Chroma Technology, Brattleboro, VT, USA). Images were processed and analyzed with the use of IMAGE PRO Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
Staging of orthotopic mouse model of human ovarian cancer. Whole-body fluorescence imaging was performed on tumor-bearing mice weekly after tumor implantation to observe tumor growth and metastasis. Each week, three mice were sacrificed for open fluorescence imaging and then primary tumor and metastases were collected for histological examination. An experienced gynecological pathologist performed the histological evaluation. Three stages of tumor progression in mice were classified with reference to the criteria of the International Federation of Gynecologists and Obstetricians (FIGO) (33, 34). Stage I tumors were those growing in the primary site and not outside the capsule of the ovary; Stage II tumors were those growing in the primary site and through the capsule of the ovary, but had not spread to surrounding tissues; Stage III tumors were those growing in multiple sites and which had metastasized to other pelvic tissues and abdominal lymph nodes.
Treatment. Extracts from AM and CW used in the study were supplied by the Nanjing University of Chinese Medicine and kept at 4°C until use. AM and CW extracts were dissolved and diluted with distilled water before administration.
Treatment of Stage I, II, and III tumors was initiated on the day determined by staging of the tumor model. For the treatments at each stage, 56 mice were randomized into seven groups of eight mice each after tumor size and progression were confirmed by fluorescence imaging for the corresponding stage. The mice at each stage were treated with the same regimen. Group 1 served as the negative control and received daily saline (NS) via oral gavage at the same dosing volume as the treated animals. Group 2 served as the positive control and received cisplatinum (CDDP) i.p. at a dose of 2 mg/kg every three days. Group 3 received AM, via oral gavage, at a dose of 9120 mg/kg. Group 4 received CW, via oral gavage, at a dose of 4560 mg/kg. Group 5 received the combination of AM and CW, via oral gavage, at a low dose (AM, 2280 mg/kg; CW, 1140 mg/kg). Group 6 received the combination, via oral gavage, at a medium dose (AM, 4560 mg/kg; CW 2280 mg/kg). Group 7 received the combination, via oral gavage, at a high dose (AM, 9120 mg/kg; CW, 4560 mg/kg). Dosing for AM and CW was performed daily until the end of the study. Each animal was examined daily for clinical signs during the treatment period. The body weight for each animal was measured twice a week to monitor toxicity. At the end of the study, tumors were dissected and final tumor weights were determined for each animal.
Effect of Astragalus membranaceus (AM), Curcuma wenyujin (CW), normal saline (NS), cisplatinum (CDDP), and low- (L), medium- (M), or high-dose (H) AM+CW on tumor matrix metalloproteinase 2 (MMP-2), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), B-cell lymphoma 2 (Bcl-2) and cyclooxygenase 2 (Cox-2). Protein expression was analyzed by immunohistochemical staining and quantitated by average optical density (AOD). A: Stage I tumors; B: Stage II tumors; C: Stage III tumors. *p<0.05, when compared with untreated control; #p<0.05, when compared with CDDP-treated and other AM- and CW-treated mice.
Immunohistochemistry. Immunohistochemistry was used to detect protein expression of matrix metalloproteinase 2 (MMP-2), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), B-cell lymphoma 2 (Bcl-2) and cyclooxygenase 2 (Cox-2) in the tumor tissue samples. The tissue sections were incubated with primary antibodies against MMP-2, VEGF, FGF-2, Bcl-2 and Cox-2 (BD Biosciences, San Diego, CA, USA), respectively, overnight at 4°C after permeabilization with a solution of 0.1% sodium citrate and 0.1%Triton X100 and blocking with 10% rabbit serum. After washing in phosphate buffered saline, the slices were incubated with horseradish peroxidase-labeled secondary antibody (1:200; Maixin BioTech Co., Ltd., Fuzhou, China) for 30 min at room temperature. After color development using diaminobenzidine (Maixin BioTech Co., Ltd), the slices were counterstained with hematoxylin and mounted with a neutral resin medium. The slides were viewed at ×400 magnification and positive cells were recognized by the appearance of brown staining. The expression level was quantified by the average optical density (AOD) of the positive cells in five fields per sample with ImagePro Plus 6.0 software. (Media Cybernetics, Silver Spring, MD, USA).
Reverse transcription-polymerase chain reaction (RT-PCR) analysis. RT-PCR was used to analyze the mRNA expression of MMP-2, Bcl-2 and FGF-2 in the tumor tissue samples. Total RNA extraction and purification were performed with an RNA extraction kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. The MMP-2, Bcl-2 and FGF-2 primers were synthesized by GenScript (Nanjing, China). The sequences of the MMP-2, Bcl-2, FGF-2 and GAPDH primers were as follows: MMP-2: Sense primer 5’-GGGGGATCCGCCTCCGAAACCATGAACTT-3’, antisense 5’-CCCGAATTCTCCTGGTGAGAGATCTGGTT-3’; Bcl-2: sense 5’-GGGGGATCCGCCTCCGAAACCATGAACTT-3’, antisense 5’-CCCGAATTCTCCTGGTGAGAGATCTGGTT-3’; FGF-2: sense 5’-GGGGGATCCGCCTCCGAAACCATGAACTT-3’, antisense 5’ CCC GAATTCTCCTGGTGAGAGATCTGGTT-3’; GAPDH: sense 5’-ATC ATCCCTGCCTCTACTGG-3’, antisense 5’-GTCAGGTCCACCACT GACAC-3’. RT-PCR was performed with an Access RT-PCR kit (Promega) according to the manufacturer's instructions. Each reaction contained 10 μl 5 × AMV/Tfl reaction buffer, 1 μl dNTP mix, 1 μl Tfi DNA polymerase, 1 μl AMV reverse transcriptase, 3 μl 25 mmol/l magnesium sulfate, 0.5 μg total RNA, 50 pmol of each of MMP-2, Bcl-2, FGF-2 and GAPHD primers. Reaction conditions for reverse transcription were: 48°C for 45 min for reverse transcription; 94°C incubation for 2 min to denature hybridized RNA/cDNA and inactivate AMV reverse transcriptase. Reaction conditions for amplification of target genes were 40 cycles of denaturing at 94°C for 30 s; annealing at 55°C for 30 s; extension at 68°C for 90 s; and final extension at 68°C for 7 min. PCR products were analyzed on 2% agarose gels and stained with ethidium bromide. Gels were scanned and images were analyzed using UNSCANIT software (SilK Scientific, Orem, UT, USA). MMP-2, Bcl-2-FGF-2, and GAPHD expression ratios were calculated.
Statistical analysis. Data are expressed as means ± SD, and were analyzed using SPSS16.0 software (SPSS inc., Chicago, Illinois, USA). Two or multiple group comparisons were performed using the Student's t-test or ANOVA. Fisher's exact test was used to compare the difference in the incidence of metastasis. A value of p≤0.05 was regarded as statistically significant.
Results
Staging of orthotopic nude mouse model of human ovarian cancer. As shown in Figure 1, the tumor at day 24 after tumor implantation met the criteria for Stage I, in which tumor was found only at the primary site and within the intact capsule of the ovary. The average tumor volume was approximately 70 mm3. The tumor at day 53 after implantation met the criteria for Stage II, in which tumor was found only at the primary site with invasion to surrounding fat tissues. The average tumor volume was approximately 250 mm3. The tumor at day 78 after tumor implantation met the criteria for Stage III, in which tumor was found in multiple sites and had metastasized to other pelvic tissues and abdominal lymph nodes.
Effect of Astragalus membranaceus (AM), Curcuma wenyujin (CW), normal saline (NS), cisplatinum (CDDP), AM, CW, or low- (L), medium- (M), or high (H)-dose AM+CW on tumor matrix metalloproteinase 2 (MMP-2), fibroblast growth factor 2 (FGF-2), and B-cell lymphoma 2 (Bcl-2) mRNA expression. Expression of MMP-2, FGF-2 and Bcl-2 mRNA was analyzed with the reverse transcription-polymerase chain reaction assay and quantitated by relative mRNA expression. A: Stage I tumors, B: Stage II tumors; C: Stage III tumors. *p<0.05 when compared with untreated controls; #p<0.05, when compared with cisplatinum (CDDP)-treated mice.
Effect of AM and CW on primary tumor growth. The Stage I study was initiated on day 24 after tumor implantation. As shown in Figure 2A and B, tumor growth was significantly reduced by high-dose AM plus CW, and CDDP compared to the untreated control at all time points in the course of treatment (p<0.05). Efficacy of AM and medium-dose AM+CW were observed only at day 41 after tumor implantation (p<0.05). As shown in Figure 2C, final tumor weights were significantly reduced compared to the untreated control; by CDDP; AM; medium-dose AM plus CW; and high-dose AM plus CW (p<0.05). High-dose AM plus CW had significantly more efficacy than other treatment groups with regard to tumor volume and final tumor weight (p<0.05). No significant tumor volume and final weight reduction were found with high-dose CW or low-dose AM plus CW compared to the untreated control (p>0.05).
None of the CDDP-, AM-, CW- and combination-treated groups showed significant reduction of tumor volume and final weight as compared to untreated control in Stage II (p>0.05) (Figure 3), or Stage III tumors (p>0.05) (Figure 4).
Effect of AM and CW on metastasis. Fewer total metastases were observed in all CDDP-, AM-, CW- and combination-treated mice compared to untreated controls in Stage I and III (Table I) tumors at autopsy, although statistical significance was not achieved (p>0.05).
Effect of AM and CW on the expression of tumor angiogenesis and apoptosis-related genes. In Stage I tumors, the protein expression of MMP-2, FGF-2, VEGF and Cox-2 was significantly reduced in mice treated with AM, CW, and all doses of AM plus CW, compared to untreated controls (p<0.05). CDDP-treated mice showed significantly reduced MMP-2, VEGF and Cox-2 protein expression compared to untreated control mice (p<0.05). MMP-2, FGF-2, VEGF, and Cox-2 protein expression levels were more greatly reduced in mice treated with AM plus CW than other treatment groups (p<0.05). Bcl-2 protein expression in the tumor was not affected in any of the treated groups (p>0.05) (Figure 5).
In Stage II tumors, only VEGF and Cox-2 protein expression was significantly reduced in treated groups compared to untreated controls (p<0.05). MMP-2, FGF-2, and Bcl-2 protein expression was not affected in any treated group (p>0.05) (Figure 5).
Effect of Astragalus membranaceus (AM) and Curcuma wenyujin (CW) on mouse body weight in the orthotopic human HO-8910-RFP ovarian cancer mouse model. A: Stage I tumors; B: Stage II tumors; C: Stage III tumors. No significant body weight loss was observed in any AM-and CW-treated mice.
In Stage III tumors, only FGF-2 and Bcl-2 protein expression was significantly reduced in treated groups compared to untreated controls (p<0.05). MMP-2 expression was only significantly reduced in the three AM and CW combination groups compared to untreated controls (p<0.05). Cox-2 and VEGF protein expression was not affected in any treated group (p>0.05) (Figure 5).
As shown in Figure 6, all treatments of Stage I, II, and III tumors significantly down-regulated the expression of MMP-2, Bcl-2 and FGF-2 mRNA compared with untreated controls (p<0.05). The expression of MMP-2, Bcl-2 and FGF-2 mRNA was significantly reduced by AM, CW, medium-dose AM plus CW, and high-dose AM plus CW more than CDDP (p<0.05).
Body weight and toxicity. Clinical observation and body weight measurement of animals during the study were performed to assess toxicity of AM and CW treatments. No physical or behavioral signs that indicated adverse effects due to the treatments were observed in any treatment group in mice with Stage I, II, and III tumors. As shown in Figure 7, stable body weight in all treated groups indicated no obvious toxicity.
Discussion
This study is the first to evaluate the efficacy of AM and CW in different stages of ovarian cancer in an orthotopic tumor mouse model. We demonstrated efficacy of the AM and CW combination in Stage I ovarian tumors. Suppression of angiogenesis- and apoptosis-related genes such as FGF-2, VEGF, MMP-2 and Cox-2 may be one of the mechanisms involved in the AM- and CW-induced inhibition of growth in the Stage I ovarian tumors. As the tumors progressed, they became treatment-resistant, similar to the clinical situation, further demonstrating the utility of the model and the need for agents active in advanced-stage ovarian cancer, such as bacterial therapy (3).
The present study is another example of the use of orthotopic mouse models and fluorescent proteins to determine the efficacy of TCM herbal mixtures (35-39).
Such studies with clinically-relevant mouse models with facile imaging capability are an important step in establishing the scientific basis of TCM.
Acknowledgements
This work was supported by grants from National Natural Science Foundation of China 81073072 and 81373990 and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
Footnotes
Conflicts of Interest
None of the Authors have any conflict of interest in regard to this study.
- Received March 10, 2015.
- Revision received March 31, 2015.
- Accepted April 3, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved