Cancer Diagnosis and Therapy
Anticachectic effects of the natural herb Coptidis rhizoma and berberine on mice bearing colon 26/clone 20 adenocarcinoma
Version of Record online: 11 MAR 2002
Copyright © 2002 Wiley-Liss, Inc.
International Journal of Cancer
Volume 99, Issue 2, pages 286–291, 10 May 2002
How to Cite
Iizuka, N., Hazama, S., Yoshimura, K., Yoshino, S., Tangoku, A., Miyamoto, K., Okita, K. and Oka, M. (2002), Anticachectic effects of the natural herb Coptidis rhizoma and berberine on mice bearing colon 26/clone 20 adenocarcinoma. Int. J. Cancer, 99: 286–291. doi: 10.1002/ijc.10338
- Issue online: 11 APR 2002
- Version of Record online: 11 MAR 2002
- Manuscript Accepted: 11 JAN 2002
- Manuscript Revised: 8 JAN 2002
- Manuscript Received: 5 OCT 2001
- Ministry of Education, Science, Sports and Culture of Japan. Grant Numbers: 12671230, 13770697
- Coptidis rhizoma;
- colon 26/clone 20 cells;
We previously showed that the natural herb Coptidis rhizoma has an anticachectic effect in nude mice bearing human esophageal cancer cells. We further investigated this phenomenon by examining the anticachectic effect of C. rhizoma in syngeneic mice bearing colon 26/clone 20 carcinoma cells, which cause IL-6–related cachexia after cell injection. We evaluated nutritional parameters such as serum glucose level and wasting of adipose tissue and muscle in tumor-bearing and non-tumor-bearing mice treated with C. rhizoma (CR) supplement or a normal diet. IL-6 levels in those mice were quantified by ELISA and real-time RT-PCR. CR supplementation significantly attenuated weight loss in tumor-bearing mice without changing food intake or tumor growth. Furthermore, these mice maintained good nutritional status. IL-6 mRNA levels in tumors and spleens and IL-6 protein levels in tumors and sera were significantly lower in tumor-bearing mice treated with CR supplement than in those treated with a normal diet. CR supplementation did not affect food intake, body weight, nutritional parameters and IL-6 levels in non-tumor-bearing mice. An in vitro study showed that C. rhizoma and its major component, berberine, inhibited IL-1–induced IL-6 mRNA expression in a dose-dependent manner in colon 26/clone 20 cells. Our results showed that C. rhizoma exerts an anticachectic effect on colon 26/clone 20–transplanted mice and that its effect is associated with tumor IL-6 production. We also suggest that its effect might be due to berberine. © 2002 Wiley-Liss, Inc.
Cancer cachexia is a paraneoplastic syndrome characterized by profound weight loss, anorexia and weakness that occurs in most malignancies.1, 2 IL-6, a mediator produced by tumors or the host as a result of tumor–host interaction, is considered to play an important role in cancer-induced cachexia, suggesting that downregulation of IL-6 levels may improve cachexia or malnutrition in cancer patients.3–6 Indeed, an anti-IL-6 receptor antibody has been shown to prevent cancer-induced cachexia in a rodent model.4 Also, medroxyprogesterone acetate (MPA), a synthetic progesterone derivative, has anticachectic activity, likely due in part to the suppression of IL-6 secretion from tumor cells.7 Thus, blockade of IL-6 function might be a useful intervention for the treatment of cachectic patients. Since these patients usually have a poor quality of life, it is essential to treat them with minimal adverse effects. These findings and considerations prompted us to look for a novel agent capable of inhibiting IL-6 with little toxicity.
From the standpoint of providing mild therapy, we focused on the use of herbs in cancer treatment. In a previous study, we identified Coptidis rhizoma as an agent possessing potent antitumor activity.8, 9 We also showed that C. rhizoma significantly attenuated weight loss in nude mice bearing IL-6–producing esophageal cancer cells at a dose that does not alter tumor growth.10 However, we were unable to clarify the relation between IL-6 and the anticachectic effect of C. rhizoma in this model. Another study showed that s.c. inoculation of colon 26/clone 20 murine colon carcinoma cells into syngeneic mice causes progressive weight loss, adipose tissue and muscle wasting and other homeostasis disorders associated with cachexia.11 Since it is likely that IL-6 is one of the critical mediators in these mice bearing colon 26/clone 20 carcinoma, we used this model to examine the link between the anticachectic effect of C. rhizoma and IL-6. Samples were analyzed by real-time RT-PCR and ELISA. We measured IL-6 mRNA and protein levels in tissues of mice treated with C. rhizoma (CR) supplement or provided the normal diet.
MATERIAL AND METHODS
C. rhizoma powder was made from Coptis japonica Makino by boiling in water and spray drying at Tsumura (Tokyo, Japan).9, 10, 12 Berberine, a major component of C. rhizoma, was purchased from Wako (Osaka, Japan). Since herbal remedies in human beings are generally administered on the free demand of patients, we designed a mouse diet containing the herb or berberine that permitted daily free intake. C. rhizoma was incorporated into breeding diet F1 at a final concentration of 1% (10 mg/g) or 2% (20 mg/g), whereas berberine was added at a final concentration of 0.1% (1 mg/g) to 0.4% (4 mg/g) at Funabashi Farms (Funabashi, Japan). For the in vitro study, C. rhizoma was dissolved in distilled water at a concentration of 30 mg/ml and berberine was resuspended in DMSO a concentration of 6 mg/ml. These solutions were prepared before the in vitro study and then mixed in culture medium. The final concentration of DMSO was kept at <0.1% to avoid its inhibitory effects on the proliferation of colon 26/clone 20 cells. Mouse IL-1α was purchased from PreproTech (London, UK).
Design of animal studies
Six-week-old male BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan). Colon 26/clone 20 cells (Nippon Roche Research Center, Kamakura, Japan) were injected s.c. (1 × 106 tumor cells/animal) with a 27-gauge needle into the right lower abdominal quadrant as previously described.11 Tumor volumes were measured with a slide caliper and calculated by the following formula: (a × b2)/2, where a is the larger and b is the smaller of the 2 dimensions.13 The standard F1 breeding diet containing berberine or C. rhizoma was provided from 4 days prior to the injection of colon 26/clone 20 cells until the animals were killed. The breeding diet alone was fed to control mice. Tumor volume and body weight were measured once every 2 days after cell injection (Fig. 1a,b). Food intake was measured once every 2 days throughout the experiment and calculated as the mean of 3 mice per cage (Fig. 1c). All mice underwent cervical dislocation 14 days after cell injection and tumor, liver, spleen and serum samples were collected and stored at –80°C until use. To investigate the effects of C. rhizoma on non-tumor-bearing mice, we set up 2 control sets based on the method described by Bing et al.14 In the pair-fed group, each animal was pair-fed to match the food intake of an individual colon 26/clone 20–bearing mouse. In the freely fed group, all animals were freely fed throughout the experiment. These mice were treated with CR supplement or a normal diet. All animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Yamaguchi University School of Medicine.
Analysis of nutritional parameters
Several nutritional parameters were evaluated, as previously described.11 Carcass weight, the difference in weight between the whole body and tumor tissue, was calculated. To determine the extent of adipose tissue and muscle wasting, we weighed the left epididymal adipose tissue and the left gastrocnemius muscle, respectively. Blood glucose levels were determined by the enzyme-electrode method with glucose oxidase (GT-1640; Hoechst Marion Roussel, Tokyo, Japan) according to the manufacturer's instructions.
Quantitative analysis of IL-6 mRNA levels
The plasmid used to generate the standard curve contained the mouse IL-6 coding region inserted into the pUC119 vector (Takara, Kyoto, Japan). Nucleotide sequences were confirmed by DNA sequencing (data not shown). The plasmid was used as template DNA in the range of 103–108 copies. Total RNA from colon 26/clone 20 cells was extracted with Trizol (GIBCO, Bethesda, MD). Four microliters of total RNA (1 μg) were reverse-transcribed in 16 μl RT mix15 containing 200 units of Maloney-murine leukemia virus reverse transcriptase (GIBCO) at 42°C for 1 hr. Two microliters of cDNA solution (equivalent to the cDNA from 100 ng of initial RNA) were used for real-time PCR amplification (LightCycler System Version 3; Roche Diagnostics, Mannheim, Germany). The primers used to detect mouse IL-6 mRNA were 5′-tccagttgccttcttgggac-3′ (sense) and 5′-gtgtaattaagcctccgact tg-3′ (antisense), which yield a 140 bp product. The PCR mixture contained 2 μl master mix (LightCycler FastStart DNA Master SYBR Green I; Roche Diagnostics), 2.4 μl (4 mM) MgCl2, 1 μl (10 pmol) of each primer, 11.6 μl distilled water and 2 μl cDNA solution. Prior to amplification, the reaction mix was incubated for 10 min at 95°C. Amplification was performed in a 3-step cycle procedure consisting of 40 cycles of denaturation at 95°C for 15 sec, annealing at 55°C for 10 sec and extension at 72°C for 6 sec. After the cycling protocol, the temperature was gradually increased at a transition rate of 0.1°C/sec. The fluorescence of all reactions was monitored throughout the temperature change to detect dissociation of the PCR products and to generate a melting curve. PCR products were quantified with a Lumi-Imager F1 (Roche Diagnostics) and subsequently analyzed with LightCycler software (Roche Diagnostics). We used the mouse β-actin gene as an internal control. We constructed a plasmid containing a 426 bp mouse β-actin coding region (positions 321–746) inserted into the pUC119 vector (Takara). Our RT-PCR method was confirmed to be quantitative at molecule numbers ranging 103–108 copies for the IL-6 gene (Fig. 2a,b) and for the μ-actin gene (data not shown). PCR products for IL-6 showed a melting temperature with a single peak at 86.19°C (Fig. 2c). PCR products were separated on a 1.5% agarose gel and confirmed to be 140 bp (data not shown). Finally, IL-6 mRNA levels were calculated as relative copy numbers to 104 copies of β-actin.
Analysis of IL-6 protein levels
Tumor, liver and spleen samples were thawed, weighed quickly, placed in 1 ml PBS and homogenized for 30 sec. Homogenates were centrifuged twice at 4°C and 10,000g and aliquots of the supernatants prepared for the IL-6 assay as described previously.5 Likewise, serum samples were thawed and subjected to the IL-6 assay. IL-6 levels were measured by ELISA using the Endogen (Boston, MA) Mouse IL-6 ELISA kit.
In vitro studies
Colon 26/clone 20 carcinoma cells (Nippon Roche Research Center) were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin G and 100 μg/ml streptomycin. Cells were plated at 1 × 106/ml in 6-well plates in RPMI medium containing 10% heat-inactivated FCS and allowed to attach overnight. The medium was then changed to RPMI containing 10% FCS with or without 10 ng/ml IL-1α, followed by administration of 0–6 μg/ml berberine or 0–30 μg/ml C. rhizoma. Cells were collected after a 2 hr incubation. RNA extraction and real-time quantitative RT-PCR were performed as described above.
For the in vivo study, the data on each nutritional parameter and IL-6 levels were analyzed by Student's t-test or ANOVA with Fisher's PLSD test. Body weight, tumor volume and food intake statistics were calculated with a 1-way ANOVA for repeated measures. For the in vitro study, IL-6 mRNA levels were analyzed by ANOVA with Fisher's PLSD test. p < 0.05 was accepted as statistically significant.
Effects of berberine on cachexia
To determine the optimal dose of C. rhizoma for this model, we preliminarily evaluated the anticachectic effects of several doses of berberine, the major component of this herb. Berberine supplementation at final concentrations of 0.2% and 0.4% of the diet significantly prevented carcass weight loss and wasting of epididymal adipose tissues and gastrocnemius muscle in tumor-bearing mice without affecting tumor growth (Table I).
|Control (n = 5)||Berberine treatment|
|0.1% (n = 6)||0.2% (n = 6)||0.4% (n = 5)|
|Tumor weight (g)||0.22 ± 0.05||0.25 ± 0.04||0.25 ± 0.05||0.24 ± 0.06|
|Carcass weight (g)||18.2 ± 0.5||18.4 ± 0.6||22.2 ± 0.52||20.9 ± 1.42|
|Gastrocnemius muscle (mg)||95.0 ± 9.0||113.5 ± 9.8||140.8 ± 15.52||133.0 ± 10.42|
|Epididymal adipose tissue (mg)||24.0 ± 7.1||26.8 ± 3.8||55.3 ± 6.32||50.2 ± 11.22|
|Serum glucose level (mg/dl)||70.4 ± 15.5||75.7 ± 8.2||120.7 ± 18.73||120.0 ± 25.03|
Effects of C. rhizoma on cachexia
Berberine belongs to the protoberberine class of isoquinoline alkaloids found in a variety of plant tissues. It is present in the extract powder of C. rhizoma at a concentration of approximately 20%.12, 16 We therefore chose to use C. rhizoma extract powder at final concentrations of 1% and 2% of the diet (CR supplement). Unfortunately, 2% CR supplementation markedly decreased food intake in mice, resulting in loss of body weight even in the tumor-free state (data not shown). Ultimately, we performed our in vivo study using only 1% CR supplementation.
The effects of 1% CR supplementation on body weight, tumor size and food intake in BALB/c mice bearing colon 26/clone 20 cells are shown in Figure 1. CR supplementation significantly prevented weight loss in tumor-bearing mice without changing tumor growth or food intake (p = 0.018). Table II summarizes the nutritional status of the 6 groups on day 14 (see Material and Methods). Total food intake between days 1 and 14 was significantly less in tumor-bearing mice and pair-fed mice than in freely fed mice (p < 0.05 for all). Final body weights and weights of epididymal adipose tissues and gastrocnemius muscle were significantly lower in tumor-bearing mice and pair-fed mice than in freely fed controls (p < 0.05 for all). Additionally, tumor-bearing mice given a normal diet had poorer nutritional status than pair-fed mice. In contrast, tumor-bearing mice given CR supplement maintained good nutritional status and their nutritional status was similar to that of pair-fed mice. Thus, in comparison to the normal diet, CR supplementation significantly prevented loss of body weight, wasting of epididymal adipose tissues and gastrocnemius muscle and hypoglycemia in colon 26/clone 20–bearing mice (p < 0.05 for all).
|Pair-fed mice||Freely fed mice|
|Normal diet||CR||Normal diet||CR||Normal diet||CR|
|(n = 9)||(n = 9)||(n = 9)||(n = 9)||(n = 6)||(n = 6)|
|Total food intake (g)||36.8 ± 1.62||36.4 ± 0.82||36.5 ± 0.62||36.5 ± 0.62||52.2 ± 0.5||52.0 ± 1.0|
|Body weight (g)|
|Initial||23.4 ± 0.3||23.2 ± 0.3||23.4 ± 0.4||23.3 ± 0.7||23.3 ± 0.4||23.7 ± 0.3|
|Final||18.7 ± 0.5234||21.8 ± 0.62||21.9 ± 0.52||22.5 ± 0.42||25.8 ± 0.4||26.1 ± 0.4|
|Gastrocnemius muscle (mg)||104.8 ± 7.0234||135.6 ± 10.32||137.6 ± 9.32||140.0 ± 9.02||169.5 ± 6.3||168.8 ± 8.8|
|Epididymal adipose tissue (mg)||21.2 ± 2.0234||45.9 ± 3.72||50.3 ± 6.92||47.6 ± 3.42||270.5 ± 7.0||258.2 ± 7.2|
|Serum glucose level (mg/dl)||70.2 ± 5.5234||110.4 ± 10.72||96.3 ± 7.32||101.8 ± 6.02||199.8 ± 12.1||188.7 ± 13.8|
Effects of CR supplementation on IL-6 levels in vivo
Table III shows IL-6 mRNA and protein levels in several tissues in the 4 groups, tumor-bearing and non-tumor-bearing mice treated with CR supplement or a normal diet. IL-6 mRNA was expressed more abundantly in tumors than in liver or spleen. IL-6 mRNA levels in tumor and spleen tissues were significantly lower in tumor-bearing mice given CR supplement than in those given a normal diet (p < 0.05 for both). CR supplementation did not affect IL-6 mRNA levels in spleen tissues of non-tumor-bearing mice. There was no statistical difference in IL-6 mRNA levels in liver tissues among the 4 groups.
|Normal diet (n = 6)||CR (n = 6)||Normal diet (n = 6)||CR (n = 6)|
|IL-6 mRNA level (copies/104 copies of β-actin)|
|Tumor||2,108 ± 159||687 ± 1253||—||—|
|Liver||55 ± 8||47 ± 11||60 ± 12||61 ± 16|
|Spleen||102 ± 12||50 ± 104||69 ± 74||60 ± 104|
|IL-6 protein level|
|Serum (pg/ml)||1,113 ± 94||598 ± 1673||<51||<51|
|Tumor (ng/g tissue)||235 ± 43||106 ± 283||—||—|
|Liver (ng/g tissue)||0.38 ± 0.15||0.46 ± 0.17||0.40 ± 0.13||0.51 ± 0.17|
|Spleen (ng/g tissue)||0.80 ± 0.22||0.70 ± 0.20||0.61 ± 0.20||0.57 ± 0.15|
IL-6 protein levels in tumor samples and sera were significantly lower in tumor-bearing mice given CR supplement than in those given a normal diet (p < 0.05 for both). Serum IL-6 levels in all non-tumor-bearing mice were below the detection limit (51 pg/ml). There was no difference in IL-6 protein levels in the liver or spleen of mice among the 4 groups.
Effects of berberine and CR on IL-6 mRNA induction by IL-1α
Our in vitro study showed that IL-6 mRNA levels in colon 26/clone 20 cells increased approximately 20-fold after 2 hr incubation with 10 ng/ml IL-1α, as described previously.17 In contrast, coincubation of cells with berberine or C. rhizoma significantly inhibited the number of IL-6 mRNA copies in a dose-dependent manner (p < 0.05, Fig. 3).
The pharmacologic and molecular actions of many herbs are gradually being elucidated. It is expected that this increased scientific understanding will give rise to a new era of herbal remedies. Studies from our laboratory and others have shown that C. rhizoma, a berberine-rich herb obtained from the root of various Coptis plants, possesses antitumor and cancer-preventive properties.9, 12, 18 Previously, we found that oral administration of C. rhizoma significantly prevented weight loss in nude mice bearing human esophageal cancer cells. IL-6 is likely to be a key molecule in cancer-induced cachexia, though any IL-6 link to the anticachectic function of this herb was unclear at that time.10 In our current study, we used an IL-6–related cachexia model and discovered a significant relation between the anticachectic effects of C. rhizoma and IL-6 levels. We observed reduced IL-6 protein levels in sera and tumor tissues and decreased IL-6 mRNA levels in tumor tissues of mice given C. rhizoma. In the same model in a different study, administration of a large dose of IL-12 suppressed cachexia induction and inhibited tumor growth.19 In addition, IFN-γ prevented induction of cachexia in this model without altering IL-6 levels.19 Our finding that C. rhizoma did not affect tumor growth suggests a different underlying mechanism for the prevention of cachexia by this herb. It has been reported that colon 26 cells produce IL-6 in vivo only in response to proinflammatory cytokines secreted by infiltrated monocytes, especially IL-1.3, 20 Moreover, Fujiki et al.17 observed that colon 26/clone 20 cells produce IL-6 in response to IL-1α, suggesting altered expression of IL-1 and IL-6 due to the interaction between tumor cells and host cells. We therefore investigated whether colon 26/clone 20 cells produce IL-6 in response to IL-1α in vitro. Our data showed that coincubation of cells with berberine or C. rhizoma inhibited IL-6 mRNA expression induced by IL-1α in a dose-dependent manner. Therefore, anticachectic activity may be attributable to transcriptional inhibition of IL-6 in tumor cells after administration of this herb. Our data also suggest that berberine, the major component, contributes significantly to the total anticachectic activity of C. rhizoma. This result corroborates the previous finding that berberine directly inhibits the activity of activator protein-1, a transactivator of IL-6.21 Cahlin et al.6 showed that omission of host IL-6, but not TNF-α, IL-12 or IFN-γ, improved carcass weight loss in knockout mice bearing MCG 101 tumors. Thus, C. rhizoma may decrease IL-6 levels derived from both tumor cells and host cells.
Anti-IL-6 receptor antibody has been shown to prevent muscle atrophy in colon 26 adenocarcinoma-bearing mice.4 In agreement with this finding, we observed that administration of C. rhizoma prevented loss of gastrocnemius muscle in colon 26/clone 20–bearing mice and decreased IL-6 levels. Tisdale1 proposed that IL-6 may be a marker rather than an actual mediator of cancer cachexia since direct administration of this cytokine to experimental animals failed to induce cachexia and Hussey et al.22 observed a relation between proteolysis-inducing factor (PIF) and cachexia in colon 26/clone 20–transplanted mice. Interestingly, muscle wasting induced by PIF may be associated with elevation of prostaglandin E2 (PGE2),23 and it is known that C. rhizoma inhibits cyclooxygenase-2 activity.12 This herb might exert anticachectic action via direct interaction with PIF and PGE2-generating pathways. We also found that CR supplementation significantly prevented hypoglycemia in tumor-bearing mice compared to the normal diet. However, it remains unclear whether its prevention is due to a direct action of C. rhizoma or not. Further studies are needed to clarify whether the anticachectic function of C. rhizoma is related to other molecules, including PIF and to examine the mechanism by which CR supplementation attenuates hypoglycemia caused by tumor inoculation.
IL-6 mRNA levels in spleen were significantly lower in tumor-bearing mice given CR supplement than in those given a normal diet. However, IL-6 protein levels in spleens were not altered. The reason for this discrepancy remains unclear. However, since significantly fewer copies of IL-6 mRNA were detected in spleen than in tumor, the difference in IL-6 mRNA levels in spleens between the CR-treated and CR-untreated tumor-bearing mice might not be biologically critical. In support of this possibility, Fujiki et al.17 reported the absence of this transcript in spleen in the same model. An alternative explanation is that administration of C. rhizoma alters certain subpopulations of cells present in spleen, thereby affecting mRNA copy number and this discrepancy might be attributable in part to a difference in the sensitivity of the 2 analytical methods used. The extract of C. rhizoma inhibits glucocorticoid-induced apoptosis in thymocytes.24 Also, the extract of C. rhizoma enhanced expression of the IL-2 gene in Jurkat cells, a human T-cell line.25 Further analyses of the effects of C. rhizoma on activity of host immune cells and subpopulations of spleen cells may provide insight into the mechanisms by which this herb acts as an anticachectic agent.
Our data showed that the whole herb C. rhizoma and its purified component berberine possess anticachectic activity. It is easy to think that purified drugs kill tumor cells more effectively than do natural plant materials because of the fact that many widely used anticancer agents are derived from natural products including herbs. Stermitz et al.26 demonstrated that Berberis fremontii, a berberine-rich herb, synthesizes a potent multidrug-resistant pump inhibitor, 5′-methoxyhydnocarpin (5′-MHC): the efflux of berberine from pathogenic Staphylococcus aureus expressing the NorA multidrug-resistant pump that confers resistance to quinolones and antiseptics was inhibited completely by 5′-MHC coexisting in B. fremontii. This is a clear example of synergy between the components of a medicinal plant, suggesting that a crude herb is not necessarily a less potent drug than one of its purified components.
In conclusion, our study showed that C. rhizoma exerts an anticachectic effect on colon 26/clone 20–transplanted mice and that the effect is associated with tumor IL-6 production. We also suggest that berberine, its major component, might play a role in the prevention of cachexia. Now, evidence from large-scale clinical trials will be necessary to incorporate this herb into mainstream cancer therapies.
We acknowledge the support of Tsumura and the Institute of Laboratory Animals, Yamaguchi University School of Medicine. We also thank Nippon Roche Research Center for providing colon 26/clone 20 cells and Dr. K. Mori for helpful advice.
- 6Experimental cancer cachexia: the role of host-derived cytokines interleukin (IL)-6, IL-12, interferon-gamma and tumor necrosis factor alpha evaluated in gene knockout, tumor-bearing mice on C57 Bl background and eicosanoid-dependent cachexia. Cancer Res 2000;60: 5488–93., , , , , .
- 8The enhancement of cisplatin-induced antitumor activity by Kampo medicines in human esophageal cancer [in Japanese with English abstract]. J Trad Med 1998;15: 340–1., , , , , .
- 16Comparative studies on concentration of berberine in plasma after oral administration of Coptidis rhizoma extract, its cultured cells extract and combined use of these extracts and glycyrrhizae radix extract in rats [in Japanese with English abstract]. Yakugaku Zasshi 1993;113: 63–9., , .