Herbal compound "Songyou Yin" reinforced the ability of interferon-alfa to inhibit the enhanced metastatic potential induced by palliative resection of hepatocellular carcinoma in nude mice

Male athymic BALB/c nu/nu mice, weighing 18-20 g at 5 weeks of age, were obtained from the Shanghai Institute of Materia Medica, Chinese Academy of Science. All mice were handled according to the recommendations of the National Institutes of Health Guidelines for Care and Use of Laboratory Animals. The experimental protocol was approved by the Shanghai Medical Experimental Animal Care Committee.

At the authors' institution, a stepwise metastatic human HCC model system was established, which included a metastatic HCC model in nude mice LCI-D20 [11], an HCC cell line MHCC97 with high metastatic potential that originated from LCI-D20 tumor [12], and cell clone MHCC97H from its parent MHCC97, with a lung metastatic rate up to 100% using orthotopic inoculation [13]. Human HCC tumor models produced by MHCC97H were established in nude mice by orthotopic inoculation, as described previously [15]. The MHCC97H cells were maintained in Dulbecco modified Eagle medium (DMEM, Gibco-BRL, Gaithersburg, MD) without any antibiotic. HCC cell lines of HCCLM3 with higher invasiveness and HCC7721, HCC7402, and Hep3B with very low invasiveness were also prepared.

The Chinese herbal medicine formula "Songyou Yin" (SYY), a dietary component authorized by the Chinese State Food and Drug Administration (Grant No.G20070160), was originally designed to promote vital energy and to be considered as a nontoxic therapeutic supplement for cancer patients [15]. The mixture includes five Chinese medicinal herbal extracts, whose proportions (w/w) are as follows: Salvia miltiorrhiza Bge., 14.3%; Astragalus membranaceus Bge., 14.3%; Lycium barbarum L., 23.8%; Crataegus pinnatifida Bge., 23.8%; and Trionyx sinensis Wiegmann, 23.8% (all from China). The ethanol extract was prepared as follows: The dried and pulverized medicinal herbs were mixed together, and each batch was poached twice for a total of 3 h, then filtrated, concentrated, and soaked with ethanol for 12 h. Finally, SYY, with the level of relative density 1.36 (60°-80°C), was obtained and stored at 4°C before application in experiments. The SYY used in the in vitro and in vivo studies, with the same batch number (#060601), was produced by the Caitong Detang Chinese Traditional Medicine Pharmaceutical Factory, Shanghai, China. A 120-mg/mL sterilized SYY by two filtrations was prepared and then diluted cell culture medium or distilled water for further use. High-performance liquid chromatography (HPLC) fingerprinting of SYY and its five characteristic components was carried out by the Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences (CAS), China. The compound was diluted with distilled water for further use, which inhibits HCC cell lines MHCC97H with high invasive potential and Hep3B cells with very low invasiveness in vitro and inhibits HCC growth and metastasis in vivo [15]. Recombinant IFN-α (Sinogen, Kexing Bioproduct Company Ltd, Shenzhen, P.R. China) inhibits HCC, which may be attributable to anti-angiogenesis [14, 16].

This study comprised 120 nude mice. In the first experiment, 48 nude mice bearing HCC xenografts were randomized into two groups 14 days after orthotopic implantation (24 mice/group), including a palliative resection group of mice undergoing partial HCC resection with preservation of 2 mm tumor [17] and a sham operation group (control) of mice having only exposure of the liver but no resection. In the second experiment, another 72 nude mice (14 days after orthotopic implantation) undergoing palliative resection were randomized into four groups according to the different therapies (18 mice/group). Therapy started on day 2 after palliative resection.

Control group mice each received 0.3 mL of distilled water via the oral gavage method once a day and were injected with sterile saline water (NS, 0.1 mL, subcutaneously) daily for 5 consecutive weeks 24 hours after resection.

Each mouse in the SYY group received 0.3 mL of "Songyou Yin" (3.6 g/kg/d/mouse) via the oral gavage method [15] and NS, 0.1 mL, subcutaneously daily.

The mice in the IFN-α group received 0.3 mL of distilled water via the oral gavage method and 0.1 mL of IFN-α (7.5 × 10⁶ U/kg/d/mouse), subcutaneously daily [14].

Finally, the SYY + IFN-α group received the combined therapy with "Songyou Yin" and IFN-α as described for the groups given each agent alone.

In the first experiment, 6 mice of each group were humanely killed by cervical dislocation 35 days after palliative resection to detect pulmonary metastasis. The remaining mice (18/group) were kept alive and their lifespan was determined starting from the day of resection; the rate of prolonged life was calculated as done previously [15]. The in situ activities of membrane type 1-matrix metalloproteinase (MT1-MMP), matrix metalloproteinase 2 (MMP2), and protein levels of MMP2, tissue inhibitor of metalloproteinase 2 (TIMP2), vascular endothelial growth factor (VEGF), and endostatin in the grown tumors obtained from the implantation sites were determined.

In the second experiment, daily general observations and weekly body weights (BWs) of the mice were recorded; 6 mice from each group were humanely killed 48 hours after the final treatment. Tumor size was measured with calipers and volume was estimated by the formula V = L × H × W × 0.5236; the tumor inhibition rate was calculated [15]. The remaining 12 mice of each group were maintained on the designated therapies until death to determine their lifespan. Lung metastases, MMP2 activity, protein levels of MMP2, TIMP2, and VEGF, and microvessel density (MVD) were determined.

Serum, lung tissues, tumor tissues, and their protein extracts were harvested for in vitro and in vivo studies.

The cells of MHCC97H, HCCLM3, HCC7721, HCC7402, and Hep3B treated with serum from killed mice and cell culture medium supplemented with 10% human AB serum (control) for 72 hours were added to the upper chamber (100 μL DMEM, 5 × 10⁴ cells/well) and 600 μL conditioned medium was added to the lower chamber. The invaded cells were fixed with methanol and stained with crystal violet solution after a 24-hour incubation. The migration assay was similar to the invasion assay, only without Matrigel. The results were expressed as the number of penetrated cells under microscope at ×200 magnification on five random fields and were presented as means ± SD of three assays [13].

Paraffin blocks of 10% buffered formalin-fixed samples of tumor and lung tissues were prepared, serial sections were cut at 5 μm, and pulmonary metastatic nodules were verified with hematoxylin and eosin stain [18].

The MT1-MMP activity assay kit (Amersham Biosciences, Buckinghamshire, UK) was used to detect active endogenous MT1-MMP in tissue extracts with analytical sensitivity of 0.7 ng/mL. The resultant color was read at 405 nm using an automated microplate reader (BioTek Instrument, Winooski, VT). The concentration of active MT1-MMP in the samples was determined by interpolation from a standard curve.

MMP2 activity in tumor tissue lysate was measured via Gelatin zymography assay [15]. The MMP activity was visualized as white proteolysis bands against the gel stained with Coomassie blue. The molecular weights of these bands indicating MMP2 activity were determined by molecular weight standards (Bio-Rad Laboratories, Hercules, CA). The relative MMP activity was quantitated by scanning the zymogram photograph on a gel documentation and analysis system (Alphalmager 2000, Alpha Innotech, San Leandro, CA). The area and optical density of each band was calculated and normalized by its own internal control; the digitized data of photographs presenting MMP2 activities in the control group were considered as 100%.

Protein levels of MMP2, TIMP2, VEGF, and endostatin in tumor extracts were measured via commercial available MMP2 (R&D Systems, Minneapolis, MN), TIMP2 (Amersham Biosciences, Buckinghamshire, UK), VEGF enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN), and endostatin (Chemicon International, Temecula, CA) kits. The mean of minimum detectable dose of MMP2, TIMP2, VEGF, and endostatin is 0.16 ng/mL, 3.2 ng/mL, 5 pg/mL, and 1.95 ng/mL, respectively. Absorbance was measured at 450 nm, using a microplate spectrophotometer with the correction wavelength of 570 nm. The assays were conducted in triplicate.

MMP2 activity was quantitated with a human MMP2 Activity ELISA System (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's instructions. The plate was read at 450 nm in a SPECTRAmax 250 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA). The assays were conducted in triplicate.

MVD (using CD34 immunostaining) was counted [19]. All slides were independently assessed by two board-certified pathologists who were blinded to the experiment. Any difference in the microvessel count was resolved by consensus.

Data were analyzed by the statistical SAS software package (SAS 8.2) using ANOVAs, Student t test, and Kaplan-Meier method (log-rank test). All continuous variables were expressed as means ± SD or means ± SE. Statistical significance was set at P < 0.05.

Increased pulmonary metastases were found in the palliative resection group (Figure 1A, left) as compared to the control (Figure 1A, right). The number of lung metastatic nodules was 14.3 ± 4.7 versus 8.7 ± 3.6 (P < 0.05, Figure 1B). However, the lifespan was longer in the palliative resection group compared with that in the control group (60.8 ± 2.7 d versus 51.3 ± 1.4 d, means ± SE, P < 0.05).

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We found that cells (MHCC97H) treated with serum from the palliative resection group (Figure 1C, left) presented the most invasive potential through Matrigel as compared to cells in controls (Figure 1C, right), with invasive ability increased by 25.7% (46.9/37.3, 1.3-fold, P < 0.05, Figure 1D), which was intervalidated by several HCC cell lines (HCCLM3, HCC7721, HCC7402, and Hep3B). However, no significant differences were found in migration capacity of treated cells among groups.

Gelatin zymography assays clearly showed that tumor MMP2 activity was stronger in the palliative resection group (Figure 2A1); the normalized optical density of the band presenting MMP2 activity was significantly elevated (1.4-fold, P < 0.01, Figure 2A2), in accord with up-regulation of the MMP2 protein level in the palliative resection group, that is, 73.7 ± 8.0 ng/mg versus 57.9 ± 10.1 ng/mg (P < 0.01, Figure 2A3). TIMP2 was down-regulated with reductions in TIMP2 levels to 15.6% of control levels (P < 0.05, Figure 2B). Tumor VEGF levels were also up-regulated by palliative resection, being 185.7 ± 13.6 pg/mg versus 164.5 ± 12.5 pg/mg (P < 0.05, Figure 2C). However, the alteration of activated tumor MT1-MMP and endostatin levels did not reach statistical significance between the two groups.

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SYY inhibited the BW loss, IFN-α was not obviously related to the BW loss, but the combined therapy noticeably minimized the BW loss, with the maximum statistical difference of BW among groups observed at the fifth week after treatment (Figure 3A). Tumor volumes in the control, SYY, IFN-α, and SYY + IFN-α groups were 1205.2 ± 581.3 mm³, 724.9 ± 337.6 mm³, 507.6 ± 367.0 mm³, and 245.3 ± 181.2 mm³, respectively (P < 0.001, for the SYY + IFN-α group compared with control, Figure 3B1,2), with tumor inhibition rates of 39.9% (SYY group), 57.9% (IFN-α group), and 79.6% (SYY + IFN-α group). At the end of the fifth week, the mean number of lung metastatic nodules was reduced by 12.3%, 46.4%, and 67.4%, respectively, compared to the control (Figure 3C). The lifespan was significantly extended in treatment groups (Figure 3D), and the rate of prolonged life was 42.2% in the SYY + IFN-α group.

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Our data revealed that both tumor MMP2 activity (Figure 4A1) and MMP2 (Figure 4A2) and VEGF (Figure 4C) protein levels were significantly down-regulated in treatment groups, whereas TIMP2 (Figure 4B) levels were up-regulated when compared to the controls. According to the inhibitory effects of SYY and IFN-α, we found that SYY was more effective on MMP2 (Figure 4A1,2), IFN-α was more effective on VEGF (Figure 4C), and the combined therapy was more effective than any agent alone. On the other hand, tumor MVD (Figure 4D1) decreased in the treated groups, being 34.5 ± 5.9, 23.5 ± 5.6, and 18.2 ± 8.0, respectively, as compared to control (43.6 ± 8.5, P < 0.001, Figure 4D2).

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No animal experienced therapy-related side effects, BW loss of > 10%, anemia, neutropenia, thrombocytopenia, or abnormal serum transaminases after the treatment regimen in this study (data not shown).