The Anti-tumor Effect of a Small Polypeptide

from

Coriolus versicolor (SPCV)

Mabel Mei Po Yang'. Zhinan Chen'. and John Shiu Lun Kwok

 Department of Physiology,. University of  Hong Kong, Hong Kong

Department of Pathology. Xijinq Institute of

Basic Medical Science. Xi'an. China

 (Accepted for publication June 1. 1992)  

Abstract: A new small polypeptide was isolated from the crude extraction of polysaccharide peptide of Coriolus versicolor (Cov-1 by HPLC and CIEF. It has a smaller molecular weight 10K) compared with that of PSP 100K) and was named small peptide of Coriolus versicolor. SPCV. It was found that SPCV possesses potent cytotoxic effect on human tumor cell lines of HL-60. LS 174-T. SMMU-7721. and SCG-7901. The IC₅₀ of SPCV on HL-60 was 30 ug/ml. The inhibition rates of leukemia cells and SCG-7901 were significantly higher in SPCV treated group than that in PSP and PSK groups. SPCV also has immuno-potentiating effect as it increased WBC and IgG levels. Pretreatment of SPCV for two weeks decreased the incidence of tumor mass in nude mice inoculated with tumor cells.

The mushroom has historically attracted attention as a health-oriented food not only in China and Japan but also world wide. The anti-tumor effect of various kinds of mushroom components have been noted during the last decade and much recent research in this field involves the development of new analytical techniques to study these pharma­cologically interesting materials (1,2.3,4,5,6). The most potent strain examined was Coriolus versicolor in which PSK (polysaccharide Krestin) was extracted from Basidiomycetes reported from Japan in 1965 (7.8) and PSP (polysaccharide peptide) from Cov-1 (Yun Zhi) reported from China in 1984 (9). Many experimental studies and clinical investigations of PSK (1,10) and PSP (9,11) in relation to their anti-tumor effect and especially for their potential use in cancer immunotherapy have been reported. It was found that the anti-tumor effect of PSP was more potent than that of PSK (12) effect of PSP was more potent than that of PSK . In vitro experiments showed that PSP inhibited the proliferation of P388 leukemia cells and Ehrlich ascites cells; it also inhibited the proliferation of some human tumor cell lines including SCG-7901, SPC, and SLY (4). In vivo experiments showed that PSP inhibited teh growth of murine sarcoma 180 in tumor bearing mice (13). The immuno-potentiating effect of PSP was also noted, and it was seen that  PSP increased the thymus weight and the serum C3 and IgG content of tumor bearing mice (14). Furthermore, PSP promoted lymphocyte proliferation and increased the production of IL-2 and interferon (INF) (15). A clinical study at the Shanghai Medical University involving 151 cases of various kinds of cancer patients who were treated with PSP. found noticeable and remarkable anti-cancer effect without toxicity to the body (11). Furthermore, PSP was of notable value in maintaining or even raising white blood cell counts, it also lessened the side effects of chemotherapy and radiotherapy, increased appetite, and relieved pain cancer patients (11) Since the PSP used in these studies was in crude extracts form.  further purification of PSP are needed.  The mechanism of the anti-tumor effect of PSP is not clear and needs further investigation. . In vitro experiments showed that PSP inhibited the proliferation of P388 leukemia cells and Ehrlich ascites cells; it also inhibited the proliferation of some human tumor cell lines including SCG-7901, SPC, and SLY (4). In vivo experiments showed that PSP inhibited teh growth of murine sarcoma 180 in tumor bearing mice (13). The immuno-potentiating effect of PSP was also noted, and it was seen that  PSP increased the thymus weight and the serum C3 and IgG content of tumor bearing mice (14). Furthermore, PSP promoted lymphocyte proliferation and increased the production of IL-2 and interferon (INF) (15). A clinical study at the Shanghai Medical University involving 151 cases of various kinds of cancer patients who were treated with PSP. found noticeable and remarkable anti-cancer effect without toxicity to the body (11). Furthermore, PSP was of notable value in maintaining or even raising white blood cell counts, it also lessened the side effects of chemotherapy and radiotherapy, increased appetite, and relieved pain cancer patients (11) Since the PSP used in these studies was in crude extracts form.  further purification of PSP are needed.  The mechanism of the anti-tumor effect of PSP is not clear and needs further investigation.

In our laboratory a new small polypeptide was isolated from the crude PSP by high performance liquid chromatography (HPLC) and capillary is electrophoresis focusing (CIEF). This biomolecule was proved to have a molecular weight of 10K (Mr) named Small Polypeptide of Coriolus Versicolor (SPCV). In our present studies we found that SPCV possesses more potent antitumor effect than the crude extraction of PSP in which a polysaccharide peptide with high molecular weight about 100 K (Mr)  from PSP was earlier reported (16).

 In vitro and in vivo evaluation

Table 1 shows that SPCV, given in a dose of 150 ug/ml to HL-60. (human leukemia cell). significantly inhibited the growth of such tumor cells with an inhibitory rate of 88.2% to 9817C on 3H-TdR incorporation after 24 hrs to 96 hrs incubation respectively. The ICSO was 30 ug/ ml which was significantly smaller than that of PSP and PSK. Thus. SPCV has more potent cytotoxicity on leukemia cell than that of PSP and PSK.

 Table 1. The inhibitory effect of SPCV on HL-60 (human leukemia cell)

Table II Cytotoxicity of SPCV on different tumor cells

• Cell were incubated with SPCV for 48 hrs.

• IC₃₀, IC₅₀, IC₉₀ were determined by ³H-TdR incorporation.

• *ug/ml

The cytotoxic effect of SPCV on several tumor cell lines was also examined and the results are shown in Table 11. It was shown that SPCV had strong inhibitory effect on the growth of HL60 (human leukemia cell), LS 174-T (colon cancer cell), SMMU-7721 (human hepatoma cell) and SCG-7901 (human stomach cancer cell). However, it had no significant inhibitory effect on the CNE-2. NPC cell.

The cytotoxic effect on SCG-7901 and leukemia cell were also compared between SPCV and other crude extractions of PSP and PSK. Table III and IV show that SPCV has marked effect on the inhibition of the growth of such tumor cells compared with the other two drugs.

 Table III. The inhibitory effect of SPCV, PSP and PSK on SCG-7901 cell

* Data obtained from Li and Xu 1987 (12). 

Table IV. The inhibitory effect or SPCV, PSP, and PSK on leukemia cell

• HL-60 cells were cultured with different concentration of drugs

• for 48 hrs. Cytotoxic activities of the drugs were assessed by ³ H-TdR incorporation for 24 hrs.

• *Data obtained from the report of Shanghai Teachers University (17).

When SPCV was injected intraperitoneally to nude mice in a dose of 2 mg daily for 10-15 days there was a marked increase in white blood cell counts, with an increase in neutrophil percentage and decrease in lymphocyte percentage at two weeks after treatment and these levels returned to normal values four weeks after treatment (Table V). Serum immunoglobulin G also increased two fold after SPCV treatment (Table VI). The increase in WBC and serum IgG represents an increase in the immune response to SPCV treatment. 

 Pretreatment of SPCV to nude mice inoculated with human tumor cells (one group inoculated with SMMU-7721 and another group inoculated with LS I 74-T). SPCV. from 30 ug to 1000 ug, was administered intraperitoneally daily for two weeks before tumor cell inoculation. The incidence of tumor mass was completely inhibited or suppressed as only one small mass of tumor tissue occurred in the group pretreated with smaller dose of SPCV (50 ug daily for two weeks)(Fig.4). Thus, the effect of pretreatment of SPCV was much better than that of the groups when treatment was given at the same time of tumor inoculation or after inoculation.

Herbal medicines have been widely used in China to treat cancer patients either in combination with western treatment or used alone. It is known that natural plants are generally less toxic to the patient. Our laboratory isolated a new small polypeptide (SPCV) from the crude extraction of Coriolus versicolor. It has relatively low toxic effect while possessing more potent antitumor effect than that of PSP. PSK and others. Our isolated SPCV has a smaller molecular weight (MW. 10K). thus, it is entirely different from the extraction of PSP (MW. 100K) polysaccharide peptide which has a large molecular weight about ten times bigger than SPCV.

The tumor cell inhibitory rate of SPCV was much higher than that of PSP and PSK. It inhibited the growth of human leukemia cell, colon cancer cell. hepatoma cell and stomach cancer cell. However, it has less cytotoxicity to human normal cell, normal liver cells and fetal lung cells (unpublished data).

SPCV also possesses an immuno-potentiating effect as it increased white blood cell counts and serum IgG levels. It also increased the organ weight of liver, spleen and thymus (unpublished data). These effects were also similar to that of PSP and PSK as their function was termed as biological response modifier (BMR). 

An ideal anti-cancer drug would be one that could directly destroy the cancer cells and indirectly stimulate the immune system activity while having less toxic effect on the body. Fortunately, SPCV possesses all of the above actions and will no doubt be developed as a new anti-cancer drug.