Combination Efficacy of Astragalus membranaceus and Curcuma wenyujin at Different Stages of Tumor Progression in an Imageable Orthotopic Nude Mouse Model of Metastatic Human Ovarian Cancer Expressing Red Fluorescent Protein

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).

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Figure 4.

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.

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Figure 5.

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.