Experimental Study of Cytobiology on Antitumor of Moxibustion and Immunomodulators

by Zhao Cuiying, Chen Yunfei, Zhao Jiazeng, Chen Hanping, Zhang Yingying, and Hong Xian

(Shanghai Research institute of Acupuncture and Meridian, Shanghai 200030, China)

Abstract: In the study, the antitumor effect was observed by employing HAC-tumor-bearing mice treated with direct moxibustion on point Guanyuan(CV 4 ) (Group M), subcutaneous administration of liposome encapsulated immunomodulators called IMC(Group IMC), and combination of these two methods (Group M + IMC). Parameters reflecting biological characteristics of tumor cells, including 5 kinds of lectins, mitotic cycle, expression of C-erbB-2 oncogene and counts of AgNORs were further investigated. The results showed that treatment with combination of moxibustion and IMC could significantly lower three lectins (ConA, LCA, RCA) among these five lectins (BSL, ConA, LCA,RCA, WGA), significantly reduce the expression of C-erbB-2 oncogene, the counts of AgNORs and the percentage of phase S in HAC tumor cells (compared with Group IMC). Moxibustion or IMC alone did render a certain degree of influence on the above-mentioned parameters, although most of changes were not statistically significant. The above-mentioned results indicated that the antitumor efficacy achieved by treatment with combination of moxibustion and IMC was mainly through its influence on biological characteristics of the tumor cells, namely, its reducing effect on DNA synthesis or on the proliferating rate of tumor cells and its influence on other biological characteristics of tumor cells.

Key Words: Moxibustion, Immunomodulation, Cytobiology

In the past, most of studies on the mechanism of antitumor action of moxibustion and acupuncture usually paid less attention to the influence of cytobiology. As a matter of fact, the development of cancer in host is a rather complicated biological process. In one hand, it is depended on biological character of tumor cell itself, in the other hand, it has a close relationship with the action or reaction between host and tumor, reflecting both sides of struggle statement between the vital energy and pathogenic factors. As the action of acupuncture and moxibustion shows synchronous regulation with multiways, multi-segment and multi-layers. Therefore in addition to studies on the side of immunology, it is relatively important to further investigate the influence of cytobiology which would promote the efficacy of antitumor effect and popularize its clinical practice.

Materials and Methods

Animals: Female C57BL/6 mice were obtained from the small animal section, Chinese Academy of Sciences (Shanghai ). They were maintained in pathogen-free conditions and were used at age 6-8 weeks.

Tumors: The HAC tumors are MCA-induced ascites carcinoma of C57BL/6 origin. These tumors were generated in our laboratory and were passaged s. c. for seven generations, at which time a cryopreserved vial from the first generation was thawed and transplanted. The single tumor cell were washed in HBSS(Biofluids, MD) counted, and diluted to a concentration of 5 x 105 cells/ml for transplantation. Treatment methods: The HAC-tumor-bearing mice treated with direct moxibustion on point Guanyuan(Group M) (two cones a day for six days, qod. one cone weighs 1. 5 mg). The second mice group treated with subcutaneous administration of liposome encapsulated immunomodulators (Group IMC). The third group treated with these combination of above two methods (Group M + IMC). The control group treated with nothing but the same dose of saline.

Flow cytometry and sorting: Freshly excised tumor tissues(0.5-2 g, wet weight) were minced into pieces smaller than 1 mm3 and washed with PBS. The mixture was poured through double Nitex sheets and harvested suspension. The supernatant was pipetted off. The pellet was placed in whirlpool mixer added with 2 ml absolute alcohol. After the pellet becoming pooled completely, stored at 4oC. Before experiment, the tumor cells should be digested with RNase (100 ug/ml, Sigma Chemical Co. St Louis. Mo) and pepsase (100 ug/ml, Difco Chemical Co. ) and stained with Ethidium Bromide. Then tested by using EPICS-I system of FCS.

Lectin receptor experiment: Preparation of samples: fresh tumor tissues were placed in 10% formalin for fixation. Formal embedding with paraffin, 5 mm serial section. Methods: adoption of enzyme immunoasssay, (ELA).

The expression of C-erbB-2 and AgNORs: The expression of C-erbB-2 refer to references with partly modifications and so did the test for AgNORs.

Results

Tumor cell cycle test: Table I demonstrates that each group show similar statement in phaseG 1, phase G 2 + M, except phase S. In comparison to group IMC, group M + IMC, and group M showed lower percentage of phase S in HAC tumor cells. The changes were statistically significant.

Lectin receptor experiment: The experiment covers 12 kinds of lectins’ binding with tumor cells. Among them, positive binding percentage range from 70%-100%, the other shows lower percentage (0-10 % ). Each group demonstrates different reaction to the binding percentage of tumor cells. The lowest binding percentage of tumor cells takes place in group M+ IMC. The expression of oncogene: According to table 3 the expression of oncogene was reduced in every group, however, the lowest one was group M + IMC. The changes were statistically significant. AgNORs in each group were similar to the expression of C-erbB-2 oncogene, that is to say, group M + IMC was the lowest one which had statistically significant changes.

Table 1. The Percentage of Tumor Cell Cycles

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Phase

Group n

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Phase G1 Phase S Phase G2+M

PI

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TB 5 33.9¡À4.1 58.3¡À 4.1 7.8¡À3.8 66.1¡À 4.1

M 6 34.2 ¡À4.3 56.9¡À4.5* 8.9¡À1.6 65.8¡À 4.3

M+IMC 5 37.9¡À6.2 53.8¡À4.7** 8.3¡À 6.8 62.1¡À 6.2

IMC 5 29.3¡À6.2 62.7¡À3.8 8.0¡À6.1 70.7¡À 6.2

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Table 2. The Changes of Lectin Receptors in Each Groups

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BSL CoA LCA RCA WGA

Group n

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£’“ + + + £’“ + + + £’“ + + + £’“ + + + £’“ + + +

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TB 10 0 7 3 0 5 5 3 2 5 1 3 6 1 3 6

M 10 2 6 2 1 4 5 6 4 0c 2 5 3 1 7 2

M+IMC 10 5 3 2 5 4 1ab 7 3 0d 5 4 1ef 0 9 1

IMC 9 3 5 1 1 6 2 5 4 0 0 7 2 3 3 3

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Table 3. The Expression of C-erbB-2 and AgNORS

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Group n C-erbB-2

AgNORS

£’“ + (granule/nuclear mean)

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TB 9 0 9 2.50¡À 0.70 (n=10)

M 10 2 8 2.60¡À 0.75(n=8)

M+IMC 10 5 5* 2.32¡À 0.59(n=9)#

IMC 9 2 7 3.04¡À 0.71(n=8)

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* Compared with group TB, P<0,05. # Compared with group IMC, P<0,05

Discussion

Animal experiment research indicated that there was a connection between the high expression of lectins receptors and hepatic carcinoma or metastasis of hepatic carcinoma. According to the result from test of HAC-bearing-mice’s 12lectins receptors, high positive expression was observed in 5 kinds of lectins receptors. The moxibustion had certain low regulation action on its positive expression. The statistically significant decrease in positive expression of 3 kinds of lectins receptors ConA, LCA, RCA among 5 lectins receptors was observed. It indicates moxibustion especially combination of moxibustion and IMC have certain influence on biological characteristics of tumor cells. Further investigation, including the expression of GerbB-2 oncogene, the counts of AgNORs and changes of tumor cell cycle, was observed that group M+IMC had lower expression of C-erbB-2 oncogene in comparison to control group, lower counts of AgNORs compared with group IMC, and the lowest percentage of phase S HAC-tumor cells C-erbB-2 plays an important role in the tumor cell’s process of development, proliferation and differentiation. It also has close relationship with the occurrence and development of tumor cells. Not only does it lead to malignant transformation but has positive correlation with malignant degree in many kinds of tumors. It has been proved that C-erbB-2oncogene had some correlation with the recurrence and metastasis of adenocarcinoma of breast and lung. The intranuclear transcription level, the number of ploidy and proliferation cycle could be observed through the technique of AgNORs. It would be helpful not only for the diagnosis of benign or malignant tumor, but also for the biological characteristics of tumor cells.

In sum, group moxibustion, especially group M+IMC, has certain antitumor effect, in that it could change the biological characters of tumor cell including speed of synthesis or proliferation, development, the degree of malignance, and other biological behaviors.

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