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Roles of three classic TCM formulae in inhibiting gastric cancer growth in mice
Journal of Traditional Chinese Medical Sciences (2017) 4, 65e70
Available online at www.sciencedirect.com
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Roles of three classic TCM formulae in inhibiting gastric cancer growth in mice Liyun Yue a, Changle Zhu a, Chong Gao a, Chong Wang a, Xinyi Chen a,*, Tongde Tian b,** a Department of Hematology and Oncology, Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing 100700, China b Integrated TCM & Western Medicine Department, Henan Cancer Hospital, Zhengzhou 450000, China
Received 2 August 2016; received in revised form 3 October 2016; accepted 7 October 2016
Available online 4 January 2017
KEYWORDS Dahuang Huanglian Xiexin Decoction; Fuzi Lizhong pills; Liushen pills; Gastric cancer; Tumor inhibition rate
Abstract Objective: To explore the effects of three classic traditional Chinese medicine (TCM) formulae, Dahuang Huanglian Xiexin Decoction (DHXD), Fuzi Lizhong pills (FLP) and Liushen pills (LSP), in suppressing gastric cancer growth in mice and provide evidence for their clinical applications. Methods: MGC-803 cells (5 106 per mouse) were injected into the subcutaneous tissues of the neck and back of nude mice. One week later, the mice were divided into three batches (9 total groups) based on tumor size and a random number table. Within each batch, the mice were subdivided into control, low- and high-dose groups for the three classic formulae. After 21d treatment, the mice were observed for their general conditions, and then sacrificed. Tumor tissues were harvested and weighed, and the tumor inhibition rate was calculated. Results: Compared with the control group, body weight in the low- and high-dose DHXD and FLP groups showed no significant change (P > .05), while the high-dose LSP group had significantly lower body weight (P Z .049). Compared with the control group, the low- and high-dose DHXD groups showed no significant inhibitory effect on xenograft tumor growth (P > .05); lowdose FLP showed an inhibitory effect on tumor growth (P Z .039), while high-dose LSP had a more significant effect (P Z .004). Conclusions: DHXD did not inhibit xenograft tumor growth in mice. In contrast, low-dose FLP had a measurable inhibitory effect, and high-dose LSP more significantly suppressed xenograft tumor growth in mice. ª 2017 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Chen), [email protected] (T. Tian). Peer review under responsibility of Beijing University of Chinese Medicine. http://dx.doi.org/10.1016/j.jtcms.2017.08.001 2095-7548/ª 2017 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Introduction Gastric cancer is a common digestive tract cancer and the leading cause of morbidity and mortality globally.1 In its early stages, gastric cancer is mainly treated by surgery; however, for more advanced gastric cancers, multidisciplinary management including chemotherapy, radiotherapy, biological therapy and traditional Chinese Medicine (TCM) are applied. In particular, TCM therapies can be used as either the main or auxiliary treatment and can relieve the clinical symptoms of gastric cancer patients, inhibit tumor growth and metastasis, regulate immune function, reduce chemotherapy-associated toxicities, prolong survival, and improve quality of life.2 Tailored TCM therapies should be applied to optimize therapeutic efficacy based on differences in the physical conditions and clinical symptoms of gastric cancer patients. However, whether such efficacies can be modeled in animal experiments remains unclear. In this study, we established xenograft mouse models of gastric cancer and compared the effects of three classic TCM formulae, Dahuang Huanglian Xiexin Decoction (DHXD), Fuzi Lizhong pills (FLP) and Liushen pills (LSP), in suppressing gastric cancer growth in mice by measuring body weights and calculating tumor inhibition rates to provide evidence for their clinical applications.
Materials Animals and tumor cells Female SPF BALB/c (nu/nu) nude mice aged 6e8 weeks and weighing [18 (2)] g were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). [Animal qualification certificate No.: 11400700132233, 11400700145018 and 11400700157398; license No.: SCXK (Beijing, China) 2012-0001]. The human gastric cancer cell line MGC-803 was purchased from the Basic Medical Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China).The Committee on the Ethics of Life Science of Zhengzhou University approved this study (approval attached in the Supplementary material).
Drug preparation The TCM formulae used in these experiments were prepared as follows: (1) DHXD: Granules manufactured by Jiangyin Tianjiang Pharmaceutical (Jiangsu, China) were used. The Chinese rhubarb (Rheum palmatum L.; batch number: 1401080) was 1 g/bag, which is equivalent to 3 g/bag for decoction pieces. The Chinese goldthread (Coptis chinensis Franch.; batch number: 1310125) was 0.5 g/bag, which is equivalent to 3 g/bag for decoction pieces. Chinese Rhubarb and Chinese goldthread were prepared into a low dose of 37.5 mg/mL and a high dose of 75.0 mg/mL at a ratio of 1:1 with sterile water, which is equivalent to 150 mg/mL and 300 mg/mL for decoction pieces, respectively. (2) FLP: Granules manufactured by Jiangyin Tianjiang Pharmaceutical were used. The root of common monkshood (Aconitum carmichaeli Debx.; batch number: 1502118) was 0.5 g/bag,
L. Yue et al. which is equivalent to 3 g/bag for decoction pieces; the ginseng (Panax ginseng C.A. Mey; batch number: 1502064) was 2.5 g/bag, which is equivalent to 10 g/bag for decoction pieces; the rhizome of largehead atractylodes (Atractylodes macrocephaia Koidz.; batch number: 1411034) was 3 g/bag, which is equivalent to 10 g/bag for decoction pieces; the dried ginger (Zingiber officinale Rosc.; batch number: 1503110) was 0.5 g/bag, which is equivalent to 3 g/bag for decoction pieces; and the licorice root (Glycyrrhiza uralensis Fisch.; batch number: 1409166) was 0.5 g/bag, which is equivalent to 3 g/bag for decoction pieces; the root of common monkshood, ginseng, the rhizome of largehead atractylodes, dried ginger and licorice root were prepared into a low dose of 118.75 mg/mL and a high dose of 237. 5 mg/ mL at a ratio of 6:10:10:9:9, respectively, with sterile water, which is equivalent to 550 mg/mL and 1100 mg/mL for decoction pieces. (3) LSP was manufactured by Ley’s Pharmaceutical (Shanghai, China; batch number: NA1001). The drug was prepared into a low dose of 1.17 mg/mL and a high dose of 2.34 mg/mL with sterile water.
Methods Modeling and grouping Three batches of mouse models were established under the same experimental conditions. After feeding for an adaptation time of one week, each mouse was injected with 5 106 MGC-803 cells (in logarithmic growth phase) into the subcutaneous tissues of neck and back. One week later, the mice were numbered based on tumor size, and then divided into three batches (9 groups) using a random number table. Batch 1 included control, low-dose DHXD and high-dose DHXD groups, with 15 mice in each group. Batch 2 included control, low-dose FLP and high-dose FLP groups, with 15 mice in each group. Batch 3 included control, low-dose LSP and high-dose LSP groups, with 10 mice in each group.
Drug administration In all three batches, animals in the control group were intragastrically fed 0.2 mL/d distilled water. Mice in the low- and high-dose DHXD groups were intragastrically fed DHXD solution at a dose of 195.00 mg/(kg$d) and 390.00 mg/(kg$d), respectively; mice in the low- and highdose FLP groups were intragastrically fed FLP solution at a dose of 617.50 mg/(kg$d) and 1235.00 mg/(kg$d), respectively; and mice in the low- and high-dose LSP groups were intragastrically fed LSP solution at a dose of 6.09 mg/(kg$d) and 12.18 mg/(kg$d), respectively. The low dose was equivalent to half the human dose, and the high dose was equivalent to the human dose. Doses were calculated according to the “equivalent dose”, with human body weight estimated as 70 kg. The animals were intragastrically fed for 21 consecutive days.
Main measures Morphologies of the mice were observed during the experiment and upon termination of the modeling. After the mice
Roles of three classic TCM formulae were sacrificed by cervical dislocation, subcutaneous tumor tissues were quickly harvested and weighed, and the tumor inhibition rate was calculated using the following formula: Tumor inhibition rate Z (average tumor weight in control group average tumor weight in therapeutic group in the same batch)/average tumor weight in control group 100%.
67 Table 1
Group
1
Group Group Group Group Group Group Group Group Group
Statistical analysis 2
Measurement data are expressed as mean (SD). Statistical analyses were performed using SPSS 21.0 software (IBM Corp., Armonk, NY). The comparison of data among multiple groups was performed using univariate analysis of variance (ANOVA). Differences between two groups were compared using t test. A P-value of less than 0.05 was considered statistically significant.
Results
Changes in body weights in each group.
Batch
3
A B C A B C A B C
No. of mice (number of xenograft tumors)
Changes in body weights [mean (SD)] (g)
10 13 12 15 15 15 10 10 10
2.16 2.29 2.16 1.63 1.21 1.74 1.03 0.72 0.51
(10) (12) (12) (14) (12) (12) (8) (8) (7)
(0.53) (0.70) (0.97) (0.61) (0.59) (0.32) (0.54) (1.55) (0.57)*
*P < .05, compared with the control group. Note: 1 Z Batch 1 (A: control group; B: low-dose DHXD group; C: high-dose DHXD group); 2 Z Batch 2 (A: control group; B: low-dose FLP group; C: high-dose FLP group); and 3 Z Batch 3 (A: control group; B: low-dose LSP group; C: high-dose LSP group).
General observations The general conditions in each group were as follows: (1) In each control group, the nude mice were active and had normal diets and water intake during the first seven days; 14 days later, the mice demonstrated varying degrees of weakness, which manifested as decreased activity along with archoptosis in some mice. (2) In the DHXD group, the diets and water intake slightly decreased during the first three days, along with decreased responsiveness and activity; later, the mice demonstrated varying degrees of weakness, along with archoptosis in some mice. (3) In the FLP group, the diets and water intake were normal during the first seven days; 14 days later, the nude mice in the lowand high-dose groups demonstrated decreased responsiveness and activity. (4) In the LSP group, the nude mice had normal responsiveness, activity, diets and water intake during the first seven days; later, the mice gradually became more excitable, with sensitive stress response, slightly increased activity and unchanged diets and water intake. Before modeling began in batch 1, five, two, and three nude mice died in the control, low-dose DHXD and high-dose DHXD groups, respectively. Autopsies showed the presence of bilateral pulmonary hilar congestion; in addition, some lungs had dissolved materials, and pleural effusion was seen in others, which might be due to improper intragastric administration.
Changes in body weights As shown in Table 1, compared with the control group, body weight in the low- and high-dose DHXD and FLP groups showed no significant difference (P > .05); conversely, the high-dose LSP group had significantly lower body weight (P Z .049).
Tumor inhibition rate Subcutaneous xenograft tumors from the three batches of model mice are shown in Figs. 1e3, and the tumor inhibition rate is shown in Table 2.
As shown in Figs. 1e3 and Table 2, low- and high-dose DHXD had no inhibitory effect on xenograft tumors (P > .05), while low-dose FLP had statistically significant inhibitory effect on xenograft tumors (P Z .039), and highdose LSP inhibited xenograft tumor growth to an even greater extent (P Z .004).
Discussion Gastric cancer is a malignant tumor of the gastric epithelial tissue. Its early clinical manifestations are often not obvious; however, in its advanced stage, patients typically suffer from upper abdominal pain, fatigue, anorexia, weight loss, metabolic disorders as well as symptoms associated with tumor expansion and metastasis. The etiologies of gastric cancer include Helicobacter pylori infection, smoking, high-salt diet and other dietary factors.3 Based on the clinical symptoms of gastric cancer, TCM named gastric cancer as wei wan tong (gastralgia), fan wei (stomach reflux), ye ge (dysphagia), fu liang (heart amassment) and ji ju (abdominal mass). Descriptions of this disease can be traced back to the Yellow Emperor’s Classic of Medicine (Huangdi Neijing), in the chapter Plain Questions (Su wen), it mentioned, “(the patients) suffer from gastralgia, which is manifested as chest pain and can affect the costal regions; in severe cases, the patients can have vomiting or diaphragm obstruction”. According to Nan Jing, “the accumulation of heart is known as fu liang, or heart amassment. It begins from the supra-umbilical area and is as large as an arm. It then reaches the epigastric area and will persist, making patients feel particularly distressed”. In Synopsis of the Golden Chamber (Jingui Yaolve), the author described the disease as follows, “the patient vomit at the evening after eating breakfast and vomit in the morning after eating supper; the overnight foods cannot be easily digested, and thus the disease is known as fan wei (stomach reflux).” According to TCM, gastric cancer is associated with improper diet, emotional disorders, qi deficiency, and external evil invasion. The
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Figure 1
L. Yue et al.
Comparing tumor sizes among groups in batch 1. A: control group; B: low-dose DHXD group; C: high-dose DHXD group.
Figure 2
Comparing tumor sizes among groups in batch 2. A: control group; B: low-dose FLP group; C: high-dose FLP group.
Figure 3
Comparing tumor sizes among groups in batch 3. A: control group; B: low-dose LSP group; C: high-dose LSP group.
main pathogenetic mechanisms include dysfunction of spleen transportation, failure of stomach qi to descend, phlegm-dampness amassment, hampering of qi movement, and endogenous toxin invasion. In Danxi Xinfa, the authors pointed out, “stomach reflux has four manifestations: blood deficiency, qi deficiency, heat, and phlegm”. The clinical diagnosis is based on TCM syndrome characteristics, which is mainly in terms of deficiency, blood stasis, phlegm and heat. Dahuang Huanglian Xiexin Decoction (DHXD) was first described in Treatise on Febrile Disease, authored by Zhang Zhongjing, a famous doctor in the Eastern Han Dynasty. The relevant texts are: “Epigastric oppression palpates soft. In patients with upward floating of pulse, DHXD can be used as the main treatment;” and “in patients with a febrile disease, recurrent sweating following major purgation often prompts epigastric oppression, which should be treated with DHXD”. In DHXD, Chinese rhubarb can remove
accumulations with purgation, clear heat-fire, eliminate blood stasis and remove obstructions in collaterals, and Chinese goldthread can clear heat, eliminate dampness, purge fire and remove toxicity. The combination of these two drugs enables clearing of heat, removing toxicity, expelling stagnation and removing obstruction in collaterals. Modern research has documented that DHXD has good therapeutic efficacy for “thermal mass” that may be a manifestation of chronic gastritis and precancerous lesions of gastric cancer.4e6 Clinical observations have also shown that gastric cancer has the clinical manifestations of “epigastric oppression”. Based on the TCM principle of “treating different diseases with the same method”, we speculate that some types of gastric cancer or certain stages in the disease course may be the indication of DHXD. Fuzi Lizhong Pill (FLP) was first recorded in Prescriptions of the Bureau of Taiping People’s Welfare Pharmacy (Taiping Huimin Heji Ju Fang). It was used to treat patients
Roles of three classic TCM formulae Table 2
Tumor inhibition rate in each group.
Batch
Group
1
Group Group Group Group Group Group Group Group Group
2
3
69
A B C A B C A B C
No. of mice (number of xenograft tumors)
Tumor weight [mean (SD)] (g)
Tumor inhibition rate (%)
10 13 12 15 15 15 10 10 10
1.12 1.22 1.28 0.48 0.33 0.45 0.46 0.40 0.20
0
(10) (12) (12) (14) (12) (12) (8) (8) (7)
(0.67) (0.71) (0.73) (0.21) (0.25) (0.29) (0.35) (0.41) (0.15)
9.20 13.83 0 30.54* 5.84 0 12.96 55.77:
*P < .05, compared with the control group. :P < .01, compared with the control group. Note: 1 Z Batch 1 (A: control group; B: low-dose DHXD group; C: high-dose DHXD group); 2 Z Batch 2 (A: control group; B: low-dose FLP group; C: high-dose FLP group); and 3 Z Batch 3 (A: control group; B: low-dose LSP group; C: high-dose LSP group).
with “dull gastralgia, deadly cold hand and foot, and abdominal pain with shortness of breath”, with “a febrile disease manifested as abdominal pain with cold syndrome”, with “persisting coldness”, and/or with “sudden diarrhea or watery diarrhea”. In FLP, the root of common monkshood can restore yang to relieve adverse effects, reinforce fire to assist yang and dispel cold to relieve pain. Dried ginger can warm the spleen and stomach to dispel cold, restore yang to remove obstructions in the collaterals, and warm the lungs to resolve fluid retention. Ginseng can nourish vitality, tonify the lungs and spleen, and tranquillize the heart and spirit. The rhizome of largehead atractylodes can replenish qi, invigorate the spleen, eliminate dampness, induce diuresis, arrest sweating, and prevent miscarriage. Finally, licorice root can tonify the spleen, replenish qi, eliminate phlegm, relieve cough, relieve pain, and clear away heat and toxic material; additionally, licorice root helps harmonize the efficacies of the various drugs. The combination of these drugs is useful for warming the spleen and stomach to dispel cold and for invigorating qi and invigorating the spleen. Modern research has demonstrated that FLP can be used to treat syndromes of deficient cold of spleen and stomach, including chronic gastritis and chronic enteritis.7,8 As FLP is indicated for treating “persisting coldness”, it may be a promising therapy for gastric cancer. LSP, also known as Lei’s Liushen Pill, was first described in Suzhou Lei Songfen Tang and is a secret formula developed by Lei Yunshang, a Suzhou doctor in Qing Dynasty. The formula consists of six ingredients: bezoar (Calculus Bovis), musk (Moschus), toad venom (Venenum Bufonis), red orpiment (Realgar), margarita (Muscovite) and borneol (Dryobalanops aromatic Gaertn. f.). It is useful for clearing heat, removing toxicity, dispersing swelling and removing knots. It can be used to treat scarlet fever, sore throat, laryngalgia/laryngeal carbuncle, unilateral/bilateral tonsillitis, pediatric furuncle, sores and carbuncles, acute mastitis, and unknown swelling and pain. Modern research has shown that LSP combined with other interventions has good efficacy in treating syndromes of toxin and blood stasis (malignancies in modern medicine); it can also help alleviate cancer-associated pain and suppress tumor angiogenesis.9,10
Based on their efficacies and indications, DHXD, FLP and LSP were all feasible treatment regimens for gastrointestinal tumors. However, to our knowledge, no study had explored their in vivo efficacy. Thus, we designed and carried out this study, from which a few major points are noteworthy. (1) In each control group, the nude mice were active and had normal diets and water intake during the first seven days; the response began to decrease on the 10th day, and 14 days later, some mice suffered from archoptosis. In the DHXD group, the diets and water intake slightly decreased over the first three days, and the animals showed decreased responsiveness and activity. After this period, diets, water intake and behavior returned to preadministration levels. After another 14 days, some mice suffered from archoptosis. In the low- and high-dose FLP groups, the nude mice had normal diets and water intake over the first seven days, but 14 days later, their activity and responsiveness decreased, and some mice suffered from archoptosis. There was no remarkable change in diets or water intake in these mice. In the LSP group, the nude mice had normal responsiveness, activity, diets and water intake over the first seven days; later, the mice gradually became more excitable, with sensitive stress response, slightly increased activity, but unchanged diets and water intake. (2) Compared with the control group, body weight in the low- and high-dose DHXD and FLP groups showed no significant change (P > .05); however the high-dose LSP group had significantly lower body weight (P < .05). (3) Furthermore, the low- and high-dose DHXD groups showed no significant inhibitory effect on xenograft tumor growth compared with the control group (P > .05), while low-dose FLP had an inhibitory effect on tumor growth (P < .05) that was increased by high-dose LSP treatment (P < .01). Thus, we conclude that: (1) DHXD has efficacies for clearing heat, removing toxicity, expelling stagnation, and removing obstruction in the collaterals, which enables it to treat the excess syndrome of epigastric oppression. However, when it is used to treat a syndrome of deficiency and excess (e.g. gastric cancer), it does not meet the principle of “achieving agreement between formula and syndrome”. Furthermore, due to its coldness and bitterness, DHXD can damage the spleen and stomach, and thus should be used
70 with caution in clinical settings. (2) FLP has efficacies for warming the spleen and stomach for dispelling cold and invigorating qi and the spleen. FLP is mainly used to treat symptoms including cold and painful stomach and abdomen, diarrhea/vomiting, and cold extremities. Lowdose FLP had an inhibitory effect on xenograft tumors in mice, whereas high-dose FLP has no such effect. No study has confirmed a therapeutic effect of FLP in gastric cancer, and further studies are required. (3) LSP is useful for clearing heat, removing toxicity, dispersing swelling, and removing knots. Clinically it is used to treat sore throat and carbuncles/furuncles. High-dose LSP can remarkably inhibit xenograft tumors in mice, which may be explained by the synergistic effects of its ingredients, which include bezoar, musk, toad venom, and red orpiment; however, whether it has a supportive role in gastric cancer treatment remains unclear. Thus, treating gastric cancer with these three formulae should be based on patient symptoms and signs. Our findings could not confirm a role for these three classic TCM formulae in treating gastric cancer due to the following considerations: First, xenograft tumors in animal models are an artificial system without immune impact; compared with tumorigenesis in humans, there are many differences in terms of immune effects and metabolism. Second, the tumor-inhibiting effects in animals cannot fully replicate that in humans. A key problem is that animals lack TCM syndromes, and thus the formula-syndrome/drugsyndrome theories cannot accurately be put into practice. Third, due to differences in factors including metabolism, immunity, and emotion, humans are different from animals in terms of drug sensitivity. An “equivalent” dose in humans may not be able to achieve the “equivalent” objective in animal models. Forth, the duration of drug use in experimental animals is remarkably shorter than in humans, and longer periods of drug use in animals may yield different outcomes. Therefore, further studies are warranted to validate our conclusions.
Funding This study is funded by National Natural Science Foundation of China (No: 81373879).
Authors contributions Tongde Tian conceived and designed the study. Xinyi Chen helped to modify the plan. Liyun Yue and Changle Zhu established the animal model and performed animal administration and data collection. Chong Gao and Chong Wang was contributed to collate and analyze the data.
L. Yue et al. Liyun Yue wrote the manuscript. All authors read and approved the final manuscript.
Conflicts of interest The authors declared no conflicts of interests.
Acknowledgment We should be very grateful for Xinyi Chen’s contribution to the manuscript.
Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jtcms.2017.08.001
References 1. Chen W, Zheng R, Baade PD, et al. Cancer statistics in China. CA Cancer J Clin. 2016;66(2):115e132. 2. Ma MY, Han KQ. Research progress of TCM in treating gastric cancer. China J Chin Med. 2015;30(6):777e779 [in Chinese]. 3. Wan JJ, Ma XG. Research progress of gastric etiology. Med Recapitulate. 2014;20(14):2542e2544 [in Chinese]. 4. Jia JH. Effectiveness of Dahuang Huanglian Xiexin Decoction in treating Helicobacter pylori-positive chronic gastritis. World Latest Med Inf. 2015;13, 135e135. [in Chinese]. 5. Xu SC. Effect of Dahuang Huanglian Xiexin decoction intervention in gastric juice components and tumor markers in patients with chronic atrophic gastritis with gastric precancerous lesions. Glob Tradit Chin Med. 2015;8(12):1425e1428 [in Chinese]. 6. Zhang Y. Effectiveness of Dahuang Huanglian Xiexin Decoction in treating Helicobacter pylori-positive chronic gastritis. China Health Care Nutr. 2016;26(21):335 [in Chinese]. 7. Tian XH. Fuzi Lizhong pills combined with acupuncture in treating chronic gastritis (syndrome of deficiency of spleen and stomach). China Health Care Nutr. 2016;26(3):97 [in Chinese]. 8. Zhang ZF. Fuzi Lizhong pills combined with heavy moxibustion method along meridians in treating chronic terminal enteritis. Seek Med Ask Med. 2013;11(3):169 [in Chinese]. 9. Cai JH, Shen SR, Wu LF, et al. Liushen Pills combined with oxycodone hydrochloride XR in treating moderate/severe cancer pain. Zhejiang J Integrated Tradit Chin West Med. 2015;8:744e745 [in Chinese]. 10. Zhang H, Huang LZ, Tian S, et al. Effects of various doses of Liushen Pill on microvessel density in models of esophageal cancer xenografts. Chin J Tissue Eng Res. 2015;5:766e771 [in Chinese].
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ScienceDirect journal homepage: http://www.elsevier.com/locate/jtcms
Roles of three classic TCM formulae in inhibiting gastric cancer growth in mice Liyun Yue a, Changle Zhu a, Chong Gao a, Chong Wang a, Xinyi Chen a,*, Tongde Tian b,** a Department of Hematology and Oncology, Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing 100700, China b Integrated TCM & Western Medicine Department, Henan Cancer Hospital, Zhengzhou 450000, China
Received 2 August 2016; received in revised form 3 October 2016; accepted 7 October 2016
Available online 4 January 2017
KEYWORDS Dahuang Huanglian Xiexin Decoction; Fuzi Lizhong pills; Liushen pills; Gastric cancer; Tumor inhibition rate
Abstract Objective: To explore the effects of three classic traditional Chinese medicine (TCM) formulae, Dahuang Huanglian Xiexin Decoction (DHXD), Fuzi Lizhong pills (FLP) and Liushen pills (LSP), in suppressing gastric cancer growth in mice and provide evidence for their clinical applications. Methods: MGC-803 cells (5 106 per mouse) were injected into the subcutaneous tissues of the neck and back of nude mice. One week later, the mice were divided into three batches (9 total groups) based on tumor size and a random number table. Within each batch, the mice were subdivided into control, low- and high-dose groups for the three classic formulae. After 21d treatment, the mice were observed for their general conditions, and then sacrificed. Tumor tissues were harvested and weighed, and the tumor inhibition rate was calculated. Results: Compared with the control group, body weight in the low- and high-dose DHXD and FLP groups showed no significant change (P > .05), while the high-dose LSP group had significantly lower body weight (P Z .049). Compared with the control group, the low- and high-dose DHXD groups showed no significant inhibitory effect on xenograft tumor growth (P > .05); lowdose FLP showed an inhibitory effect on tumor growth (P Z .039), while high-dose LSP had a more significant effect (P Z .004). Conclusions: DHXD did not inhibit xenograft tumor growth in mice. In contrast, low-dose FLP had a measurable inhibitory effect, and high-dose LSP more significantly suppressed xenograft tumor growth in mice. ª 2017 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Chen), [email protected] (T. Tian). Peer review under responsibility of Beijing University of Chinese Medicine. http://dx.doi.org/10.1016/j.jtcms.2017.08.001 2095-7548/ª 2017 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
66
Introduction Gastric cancer is a common digestive tract cancer and the leading cause of morbidity and mortality globally.1 In its early stages, gastric cancer is mainly treated by surgery; however, for more advanced gastric cancers, multidisciplinary management including chemotherapy, radiotherapy, biological therapy and traditional Chinese Medicine (TCM) are applied. In particular, TCM therapies can be used as either the main or auxiliary treatment and can relieve the clinical symptoms of gastric cancer patients, inhibit tumor growth and metastasis, regulate immune function, reduce chemotherapy-associated toxicities, prolong survival, and improve quality of life.2 Tailored TCM therapies should be applied to optimize therapeutic efficacy based on differences in the physical conditions and clinical symptoms of gastric cancer patients. However, whether such efficacies can be modeled in animal experiments remains unclear. In this study, we established xenograft mouse models of gastric cancer and compared the effects of three classic TCM formulae, Dahuang Huanglian Xiexin Decoction (DHXD), Fuzi Lizhong pills (FLP) and Liushen pills (LSP), in suppressing gastric cancer growth in mice by measuring body weights and calculating tumor inhibition rates to provide evidence for their clinical applications.
Materials Animals and tumor cells Female SPF BALB/c (nu/nu) nude mice aged 6e8 weeks and weighing [18 (2)] g were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). [Animal qualification certificate No.: 11400700132233, 11400700145018 and 11400700157398; license No.: SCXK (Beijing, China) 2012-0001]. The human gastric cancer cell line MGC-803 was purchased from the Basic Medical Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China).The Committee on the Ethics of Life Science of Zhengzhou University approved this study (approval attached in the Supplementary material).
Drug preparation The TCM formulae used in these experiments were prepared as follows: (1) DHXD: Granules manufactured by Jiangyin Tianjiang Pharmaceutical (Jiangsu, China) were used. The Chinese rhubarb (Rheum palmatum L.; batch number: 1401080) was 1 g/bag, which is equivalent to 3 g/bag for decoction pieces. The Chinese goldthread (Coptis chinensis Franch.; batch number: 1310125) was 0.5 g/bag, which is equivalent to 3 g/bag for decoction pieces. Chinese Rhubarb and Chinese goldthread were prepared into a low dose of 37.5 mg/mL and a high dose of 75.0 mg/mL at a ratio of 1:1 with sterile water, which is equivalent to 150 mg/mL and 300 mg/mL for decoction pieces, respectively. (2) FLP: Granules manufactured by Jiangyin Tianjiang Pharmaceutical were used. The root of common monkshood (Aconitum carmichaeli Debx.; batch number: 1502118) was 0.5 g/bag,
L. Yue et al. which is equivalent to 3 g/bag for decoction pieces; the ginseng (Panax ginseng C.A. Mey; batch number: 1502064) was 2.5 g/bag, which is equivalent to 10 g/bag for decoction pieces; the rhizome of largehead atractylodes (Atractylodes macrocephaia Koidz.; batch number: 1411034) was 3 g/bag, which is equivalent to 10 g/bag for decoction pieces; the dried ginger (Zingiber officinale Rosc.; batch number: 1503110) was 0.5 g/bag, which is equivalent to 3 g/bag for decoction pieces; and the licorice root (Glycyrrhiza uralensis Fisch.; batch number: 1409166) was 0.5 g/bag, which is equivalent to 3 g/bag for decoction pieces; the root of common monkshood, ginseng, the rhizome of largehead atractylodes, dried ginger and licorice root were prepared into a low dose of 118.75 mg/mL and a high dose of 237. 5 mg/ mL at a ratio of 6:10:10:9:9, respectively, with sterile water, which is equivalent to 550 mg/mL and 1100 mg/mL for decoction pieces. (3) LSP was manufactured by Ley’s Pharmaceutical (Shanghai, China; batch number: NA1001). The drug was prepared into a low dose of 1.17 mg/mL and a high dose of 2.34 mg/mL with sterile water.
Methods Modeling and grouping Three batches of mouse models were established under the same experimental conditions. After feeding for an adaptation time of one week, each mouse was injected with 5 106 MGC-803 cells (in logarithmic growth phase) into the subcutaneous tissues of neck and back. One week later, the mice were numbered based on tumor size, and then divided into three batches (9 groups) using a random number table. Batch 1 included control, low-dose DHXD and high-dose DHXD groups, with 15 mice in each group. Batch 2 included control, low-dose FLP and high-dose FLP groups, with 15 mice in each group. Batch 3 included control, low-dose LSP and high-dose LSP groups, with 10 mice in each group.
Drug administration In all three batches, animals in the control group were intragastrically fed 0.2 mL/d distilled water. Mice in the low- and high-dose DHXD groups were intragastrically fed DHXD solution at a dose of 195.00 mg/(kg$d) and 390.00 mg/(kg$d), respectively; mice in the low- and highdose FLP groups were intragastrically fed FLP solution at a dose of 617.50 mg/(kg$d) and 1235.00 mg/(kg$d), respectively; and mice in the low- and high-dose LSP groups were intragastrically fed LSP solution at a dose of 6.09 mg/(kg$d) and 12.18 mg/(kg$d), respectively. The low dose was equivalent to half the human dose, and the high dose was equivalent to the human dose. Doses were calculated according to the “equivalent dose”, with human body weight estimated as 70 kg. The animals were intragastrically fed for 21 consecutive days.
Main measures Morphologies of the mice were observed during the experiment and upon termination of the modeling. After the mice
Roles of three classic TCM formulae were sacrificed by cervical dislocation, subcutaneous tumor tissues were quickly harvested and weighed, and the tumor inhibition rate was calculated using the following formula: Tumor inhibition rate Z (average tumor weight in control group average tumor weight in therapeutic group in the same batch)/average tumor weight in control group 100%.
67 Table 1
Group
1
Group Group Group Group Group Group Group Group Group
Statistical analysis 2
Measurement data are expressed as mean (SD). Statistical analyses were performed using SPSS 21.0 software (IBM Corp., Armonk, NY). The comparison of data among multiple groups was performed using univariate analysis of variance (ANOVA). Differences between two groups were compared using t test. A P-value of less than 0.05 was considered statistically significant.
Results
Changes in body weights in each group.
Batch
3
A B C A B C A B C
No. of mice (number of xenograft tumors)
Changes in body weights [mean (SD)] (g)
10 13 12 15 15 15 10 10 10
2.16 2.29 2.16 1.63 1.21 1.74 1.03 0.72 0.51
(10) (12) (12) (14) (12) (12) (8) (8) (7)
(0.53) (0.70) (0.97) (0.61) (0.59) (0.32) (0.54) (1.55) (0.57)*
*P < .05, compared with the control group. Note: 1 Z Batch 1 (A: control group; B: low-dose DHXD group; C: high-dose DHXD group); 2 Z Batch 2 (A: control group; B: low-dose FLP group; C: high-dose FLP group); and 3 Z Batch 3 (A: control group; B: low-dose LSP group; C: high-dose LSP group).
General observations The general conditions in each group were as follows: (1) In each control group, the nude mice were active and had normal diets and water intake during the first seven days; 14 days later, the mice demonstrated varying degrees of weakness, which manifested as decreased activity along with archoptosis in some mice. (2) In the DHXD group, the diets and water intake slightly decreased during the first three days, along with decreased responsiveness and activity; later, the mice demonstrated varying degrees of weakness, along with archoptosis in some mice. (3) In the FLP group, the diets and water intake were normal during the first seven days; 14 days later, the nude mice in the lowand high-dose groups demonstrated decreased responsiveness and activity. (4) In the LSP group, the nude mice had normal responsiveness, activity, diets and water intake during the first seven days; later, the mice gradually became more excitable, with sensitive stress response, slightly increased activity and unchanged diets and water intake. Before modeling began in batch 1, five, two, and three nude mice died in the control, low-dose DHXD and high-dose DHXD groups, respectively. Autopsies showed the presence of bilateral pulmonary hilar congestion; in addition, some lungs had dissolved materials, and pleural effusion was seen in others, which might be due to improper intragastric administration.
Changes in body weights As shown in Table 1, compared with the control group, body weight in the low- and high-dose DHXD and FLP groups showed no significant difference (P > .05); conversely, the high-dose LSP group had significantly lower body weight (P Z .049).
Tumor inhibition rate Subcutaneous xenograft tumors from the three batches of model mice are shown in Figs. 1e3, and the tumor inhibition rate is shown in Table 2.
As shown in Figs. 1e3 and Table 2, low- and high-dose DHXD had no inhibitory effect on xenograft tumors (P > .05), while low-dose FLP had statistically significant inhibitory effect on xenograft tumors (P Z .039), and highdose LSP inhibited xenograft tumor growth to an even greater extent (P Z .004).
Discussion Gastric cancer is a malignant tumor of the gastric epithelial tissue. Its early clinical manifestations are often not obvious; however, in its advanced stage, patients typically suffer from upper abdominal pain, fatigue, anorexia, weight loss, metabolic disorders as well as symptoms associated with tumor expansion and metastasis. The etiologies of gastric cancer include Helicobacter pylori infection, smoking, high-salt diet and other dietary factors.3 Based on the clinical symptoms of gastric cancer, TCM named gastric cancer as wei wan tong (gastralgia), fan wei (stomach reflux), ye ge (dysphagia), fu liang (heart amassment) and ji ju (abdominal mass). Descriptions of this disease can be traced back to the Yellow Emperor’s Classic of Medicine (Huangdi Neijing), in the chapter Plain Questions (Su wen), it mentioned, “(the patients) suffer from gastralgia, which is manifested as chest pain and can affect the costal regions; in severe cases, the patients can have vomiting or diaphragm obstruction”. According to Nan Jing, “the accumulation of heart is known as fu liang, or heart amassment. It begins from the supra-umbilical area and is as large as an arm. It then reaches the epigastric area and will persist, making patients feel particularly distressed”. In Synopsis of the Golden Chamber (Jingui Yaolve), the author described the disease as follows, “the patient vomit at the evening after eating breakfast and vomit in the morning after eating supper; the overnight foods cannot be easily digested, and thus the disease is known as fan wei (stomach reflux).” According to TCM, gastric cancer is associated with improper diet, emotional disorders, qi deficiency, and external evil invasion. The
68
Figure 1
L. Yue et al.
Comparing tumor sizes among groups in batch 1. A: control group; B: low-dose DHXD group; C: high-dose DHXD group.
Figure 2
Comparing tumor sizes among groups in batch 2. A: control group; B: low-dose FLP group; C: high-dose FLP group.
Figure 3
Comparing tumor sizes among groups in batch 3. A: control group; B: low-dose LSP group; C: high-dose LSP group.
main pathogenetic mechanisms include dysfunction of spleen transportation, failure of stomach qi to descend, phlegm-dampness amassment, hampering of qi movement, and endogenous toxin invasion. In Danxi Xinfa, the authors pointed out, “stomach reflux has four manifestations: blood deficiency, qi deficiency, heat, and phlegm”. The clinical diagnosis is based on TCM syndrome characteristics, which is mainly in terms of deficiency, blood stasis, phlegm and heat. Dahuang Huanglian Xiexin Decoction (DHXD) was first described in Treatise on Febrile Disease, authored by Zhang Zhongjing, a famous doctor in the Eastern Han Dynasty. The relevant texts are: “Epigastric oppression palpates soft. In patients with upward floating of pulse, DHXD can be used as the main treatment;” and “in patients with a febrile disease, recurrent sweating following major purgation often prompts epigastric oppression, which should be treated with DHXD”. In DHXD, Chinese rhubarb can remove
accumulations with purgation, clear heat-fire, eliminate blood stasis and remove obstructions in collaterals, and Chinese goldthread can clear heat, eliminate dampness, purge fire and remove toxicity. The combination of these two drugs enables clearing of heat, removing toxicity, expelling stagnation and removing obstruction in collaterals. Modern research has documented that DHXD has good therapeutic efficacy for “thermal mass” that may be a manifestation of chronic gastritis and precancerous lesions of gastric cancer.4e6 Clinical observations have also shown that gastric cancer has the clinical manifestations of “epigastric oppression”. Based on the TCM principle of “treating different diseases with the same method”, we speculate that some types of gastric cancer or certain stages in the disease course may be the indication of DHXD. Fuzi Lizhong Pill (FLP) was first recorded in Prescriptions of the Bureau of Taiping People’s Welfare Pharmacy (Taiping Huimin Heji Ju Fang). It was used to treat patients
Roles of three classic TCM formulae Table 2
Tumor inhibition rate in each group.
Batch
Group
1
Group Group Group Group Group Group Group Group Group
2
3
69
A B C A B C A B C
No. of mice (number of xenograft tumors)
Tumor weight [mean (SD)] (g)
Tumor inhibition rate (%)
10 13 12 15 15 15 10 10 10
1.12 1.22 1.28 0.48 0.33 0.45 0.46 0.40 0.20
0
(10) (12) (12) (14) (12) (12) (8) (8) (7)
(0.67) (0.71) (0.73) (0.21) (0.25) (0.29) (0.35) (0.41) (0.15)
9.20 13.83 0 30.54* 5.84 0 12.96 55.77:
*P < .05, compared with the control group. :P < .01, compared with the control group. Note: 1 Z Batch 1 (A: control group; B: low-dose DHXD group; C: high-dose DHXD group); 2 Z Batch 2 (A: control group; B: low-dose FLP group; C: high-dose FLP group); and 3 Z Batch 3 (A: control group; B: low-dose LSP group; C: high-dose LSP group).
with “dull gastralgia, deadly cold hand and foot, and abdominal pain with shortness of breath”, with “a febrile disease manifested as abdominal pain with cold syndrome”, with “persisting coldness”, and/or with “sudden diarrhea or watery diarrhea”. In FLP, the root of common monkshood can restore yang to relieve adverse effects, reinforce fire to assist yang and dispel cold to relieve pain. Dried ginger can warm the spleen and stomach to dispel cold, restore yang to remove obstructions in the collaterals, and warm the lungs to resolve fluid retention. Ginseng can nourish vitality, tonify the lungs and spleen, and tranquillize the heart and spirit. The rhizome of largehead atractylodes can replenish qi, invigorate the spleen, eliminate dampness, induce diuresis, arrest sweating, and prevent miscarriage. Finally, licorice root can tonify the spleen, replenish qi, eliminate phlegm, relieve cough, relieve pain, and clear away heat and toxic material; additionally, licorice root helps harmonize the efficacies of the various drugs. The combination of these drugs is useful for warming the spleen and stomach to dispel cold and for invigorating qi and invigorating the spleen. Modern research has demonstrated that FLP can be used to treat syndromes of deficient cold of spleen and stomach, including chronic gastritis and chronic enteritis.7,8 As FLP is indicated for treating “persisting coldness”, it may be a promising therapy for gastric cancer. LSP, also known as Lei’s Liushen Pill, was first described in Suzhou Lei Songfen Tang and is a secret formula developed by Lei Yunshang, a Suzhou doctor in Qing Dynasty. The formula consists of six ingredients: bezoar (Calculus Bovis), musk (Moschus), toad venom (Venenum Bufonis), red orpiment (Realgar), margarita (Muscovite) and borneol (Dryobalanops aromatic Gaertn. f.). It is useful for clearing heat, removing toxicity, dispersing swelling and removing knots. It can be used to treat scarlet fever, sore throat, laryngalgia/laryngeal carbuncle, unilateral/bilateral tonsillitis, pediatric furuncle, sores and carbuncles, acute mastitis, and unknown swelling and pain. Modern research has shown that LSP combined with other interventions has good efficacy in treating syndromes of toxin and blood stasis (malignancies in modern medicine); it can also help alleviate cancer-associated pain and suppress tumor angiogenesis.9,10
Based on their efficacies and indications, DHXD, FLP and LSP were all feasible treatment regimens for gastrointestinal tumors. However, to our knowledge, no study had explored their in vivo efficacy. Thus, we designed and carried out this study, from which a few major points are noteworthy. (1) In each control group, the nude mice were active and had normal diets and water intake during the first seven days; the response began to decrease on the 10th day, and 14 days later, some mice suffered from archoptosis. In the DHXD group, the diets and water intake slightly decreased over the first three days, and the animals showed decreased responsiveness and activity. After this period, diets, water intake and behavior returned to preadministration levels. After another 14 days, some mice suffered from archoptosis. In the low- and high-dose FLP groups, the nude mice had normal diets and water intake over the first seven days, but 14 days later, their activity and responsiveness decreased, and some mice suffered from archoptosis. There was no remarkable change in diets or water intake in these mice. In the LSP group, the nude mice had normal responsiveness, activity, diets and water intake over the first seven days; later, the mice gradually became more excitable, with sensitive stress response, slightly increased activity, but unchanged diets and water intake. (2) Compared with the control group, body weight in the low- and high-dose DHXD and FLP groups showed no significant change (P > .05); however the high-dose LSP group had significantly lower body weight (P < .05). (3) Furthermore, the low- and high-dose DHXD groups showed no significant inhibitory effect on xenograft tumor growth compared with the control group (P > .05), while low-dose FLP had an inhibitory effect on tumor growth (P < .05) that was increased by high-dose LSP treatment (P < .01). Thus, we conclude that: (1) DHXD has efficacies for clearing heat, removing toxicity, expelling stagnation, and removing obstruction in the collaterals, which enables it to treat the excess syndrome of epigastric oppression. However, when it is used to treat a syndrome of deficiency and excess (e.g. gastric cancer), it does not meet the principle of “achieving agreement between formula and syndrome”. Furthermore, due to its coldness and bitterness, DHXD can damage the spleen and stomach, and thus should be used
70 with caution in clinical settings. (2) FLP has efficacies for warming the spleen and stomach for dispelling cold and invigorating qi and the spleen. FLP is mainly used to treat symptoms including cold and painful stomach and abdomen, diarrhea/vomiting, and cold extremities. Lowdose FLP had an inhibitory effect on xenograft tumors in mice, whereas high-dose FLP has no such effect. No study has confirmed a therapeutic effect of FLP in gastric cancer, and further studies are required. (3) LSP is useful for clearing heat, removing toxicity, dispersing swelling, and removing knots. Clinically it is used to treat sore throat and carbuncles/furuncles. High-dose LSP can remarkably inhibit xenograft tumors in mice, which may be explained by the synergistic effects of its ingredients, which include bezoar, musk, toad venom, and red orpiment; however, whether it has a supportive role in gastric cancer treatment remains unclear. Thus, treating gastric cancer with these three formulae should be based on patient symptoms and signs. Our findings could not confirm a role for these three classic TCM formulae in treating gastric cancer due to the following considerations: First, xenograft tumors in animal models are an artificial system without immune impact; compared with tumorigenesis in humans, there are many differences in terms of immune effects and metabolism. Second, the tumor-inhibiting effects in animals cannot fully replicate that in humans. A key problem is that animals lack TCM syndromes, and thus the formula-syndrome/drugsyndrome theories cannot accurately be put into practice. Third, due to differences in factors including metabolism, immunity, and emotion, humans are different from animals in terms of drug sensitivity. An “equivalent” dose in humans may not be able to achieve the “equivalent” objective in animal models. Forth, the duration of drug use in experimental animals is remarkably shorter than in humans, and longer periods of drug use in animals may yield different outcomes. Therefore, further studies are warranted to validate our conclusions.
Funding This study is funded by National Natural Science Foundation of China (No: 81373879).
Authors contributions Tongde Tian conceived and designed the study. Xinyi Chen helped to modify the plan. Liyun Yue and Changle Zhu established the animal model and performed animal administration and data collection. Chong Gao and Chong Wang was contributed to collate and analyze the data.
L. Yue et al. Liyun Yue wrote the manuscript. All authors read and approved the final manuscript.
Conflicts of interest The authors declared no conflicts of interests.
Acknowledgment We should be very grateful for Xinyi Chen’s contribution to the manuscript.
Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jtcms.2017.08.001
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