Cryoablation is a low-invasive surgical treatment for malignant tumors. It may induce an immunological response leading to the eradication of distant metastases or alternatively it might promote the growth of residual tumors. In this paper we confirm the occurrence of both phenomena and we describe the preventive effect of a protein-bound polysaccharide preparation. Metastatic liver tumors were produced in BALB/c mice by the intrasplenic inoculation of colon 26 cells and cryoablation was carried out usingliquid nitrogen ()170C) applied by a contact method. The value of combiningcryoablation with administration of the polysaccharide preparation in the prevention of growth of residual tumors was investigated. It was shown that the number of metastatic liver nodules and the size of the primary tumor at the site of inoculation in the spleen were significantly lower when the volume that was frozen was small. The production by splenocytes of the tumor necrosis factor TNF-a, interferon INF-c, and the interleukins IL-4 and IL-10 increased significantly after freezing and thawing of the tumor tissue. The polysaccharide treatment significantly reduced the production of IL-4 and IL-10 followingcryoablation; the production of TNF-a and INF-c was slightly promoted; the natural killer and cytotoxic T-cell activities of splenocytes were slightly enhanced. It was concluded that the polysaccharide preparation was beneficial by suppressing IL-4 and IL-10 production and might inhibit the growth of residual tumor that is sometimes induced by large-volume cryoablation.
RPSP, a refined polysaccharide peptide fraction isolated by fast performance liquid chromatography (FPLC) from the crude powder of total peptide-bound polysaccharides of cultivated Coriolus versicolor Cov-1 dose-dependently inhibited the proliferation of a human hepatoma cell line (HEPG2). The effective dose causing 50% inhibition following 3-day exposure to RPSP was 243 +/- 36 micrograms/ml for HEPG2. However, little or no inhibitory effects were detected in normal human foetal hepatocytes. On the other hand, in the pretreatment group, in which RPSP was administered i.p. for two weeks before sarcoma 180 inoculation in nude mice, the incidence of tumor growth was less (2 out of 5 mice) than that of the control group (all 5 mice). The tumor size of the control group was about 3-5 times bigger than that of the pretreatment group. In tumor bearing nude mice, 5 days after sarcoma 180 inoculation, i.v. administration of RPSP significantly suppressed the growth of tumor mass. The inhibition rate was 93.6% on day 13. Furthermore, administration of RPSP did not cause any pathological lesions in vital organs of rabbits such as heart, liver, spleen, lung and kidney. In conclusion, these results indicate that RPSP acts by directly suppressing tumor cell growth in vitro and the prevention of in vivo growth of tumor mass is probably mediated also via its immunomodulating effects.
Hiroshi Kobayashi, Kenichi Matsunaga,1 and Yoshiharu Oguchi
P5K, a protein-bound polysaccharide obtained from cultured mycelia of Coriolus versicolor in basidiomycetes, is a biological response modifier, diverse operations of which include an antitumor action. We have previously reviewed recent research which had demonstrated that in animals, P5K has a preventive effect on chemical carcinogen-induced, radiation induced, and spontaneously developed carcinogenesis (Kobayashi et aL, Cancer Epidemiol., Biomarkers & Prey., 2: 271-276, 1993). We now focus on the effects of PSK once the progression of carcinogenesis has begun, and review what is now known of the preventive action of PSK on cancer metastasis.
School of Pharmacy, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Abstract
Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells, but the underlying mechanism has not been fully elucidated. The present study aimed to evaluate the in vitro anti-tumor activity of a standardized aqueous ethanol extract prepared from CV on four breast cancer cell lines using MTT assay, and test whether the mechanism involves apoptosis induction and modulation of p53 and Bcl-2 protein expressions using cell death detection ELISA, p53 and Bcl-2 ELISAs respectively. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of three breast tumor cell lines, with ascending order of IC50 values: T-47D, MCF-7, MDA-MB-231, while BT-20 cells were not significantly affected. Tumoricidal activity of the CV extract was found to be comparable to a chemotherapeutic anti-cancer drug, mitomycin C. Nucleosome productions in apoptotic MDA-MB-231, MCF-7 and T-47D cells were significantly augmented in a time-dependent manner and paralleled the anti-proliferative activity of CV extract. Expression of p53 protein was significantly upregulated only in T-47D cells treated with the CV extract in a dose- and time-dependent fashion, but not in MCF-7 (except at 400 mug/ml after 16 h) and MDA-MB-231 cells. The CV extract significantly induced a dose-dependent downregulation of Bcl-2 protein expression in MCF-7 and T-47D cells, but not in MDA-MB-231 cells. These results suggested that apoptosis induction, differentially dependent of p53 and Bcl-2 expressions, might be the possible mechanism of CV extract-mediated cytotoxicity in human breast cancer cells in vitro.
PMID: 15908782 [PubMed – indexed for MEDLINE]Free Article
Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, P.R. China.
Abstract
Activation of the cell death program (apoptosis) is a strategy for the treatment of human cancer, and unfortunately a large number of drugs identified as cell cycle-specific agents for killing cancer cells are also toxic to normal cells. The present study demonstrates that the polysaccharide peptide (PSP) extracted from the Chinese medicinal mushroom, Coriolus versicolor, used in combination therapy in China, has the ability to lower the cytotoxicity of certain anti-leukemic drugs via their interaction with cell cycle-dependent and apoptotic pathways. Flow cytometry analysis demonstrated that pre-treatment of PSP (25-100 microg/ml) dose-dependently enhanced the cell cycle perturbation and apoptotic activity of doxorubicin (Doxo) and etoposide (VP-16), but not cytarabine (Ara-C) in human promyelocytic leukemia HL-60 cells. The antagonistic result from combined treatment with Ara-C and PSP may be caused by the removal of HL-60 cells in the G1-S boundary by PSP before exposure to Ara-C. A negative correlation between the increase in apoptotic cell population (pre-G1 peak) with the S-phase cell population expression (R2=0.998), the expression of cyclin E expression (R2=0.872) and caspase 3 activity (R2=0.997) suggests that PSP enhanced the apoptotic machinery of Doxo and VP-16 in a cell cycle-dependent manner and is mediated, at least in part, by the PSP-mediated modulation of the regulatory checkpoint cyclin E and caspase 3. This study is the first to describe the cell cycle mechanistic action of PSP and its interaction with other anticancer agents. Our data support the potential development of PSP as an adjuvant for leukemia treatment, but also imply the importance of understanding its interaction with individual anticancer agents.
We examined the effect of PSK, a protein-bound polysaccharide, upon in vivo metastasis and in vitro invasion of the B16-BL6 mouse melanoma cells. (1) PSK suppressed in vivo artificial and spontaneous lung metastases of B16-BL6 in C57BL/6 mice. (2) PSK in a dose-dependent fashion suppressed in vitro invasion and chemotaxis of the tumor cells using filters coated with a reconstituted basement membrane. (3) PSK had little effect on DNA synthesis in tumor cells in vitro, but suppressed tumor cell adhesion to, degradation of, and haptotaxis to components of the basement membrane. (4) PSK suppressed the binding of tumor cells to components of the basement membrane. These findings suggest that PSK may suppress metastasis through inhibition of tumor cell invasion and that this effect is the result of interactions between PSK and components of the basement membrane.
The anticancer effects of PSP purified products, PSP-A, PSP-B, PSP-C and crude product PSP-Cr and KS-2 were compared on four human tumor cell lines in vitro. It was found that the inhibition rate of cell proliferation of PSP-A was higher than that of PSP-Cr, PSP-B and PSP-C (P<0.05). On SPC cells, the inhibition rate of PSP-A at a dosage of 1000ug/ml was 62.7%, being the highest as compared with those on the other three cell lines.
The effect of the inhibition of KS-2 on Mei tumorous cells is obvious. Its inhibition rate is 62.5%.
Morphological changes were seen in all the four cell lines, especially in SPC cells after PSP-A treatment.
To evaluate the efficacy of PSK for adjuvant immuno-chemotherapy in patients who had undergone radical gastrectomy, a randomized controlled trial has been in progress in collaboration with 46 institutions in the Chubu district of Japan. A total of 262 patients were registered for this trial during the two years from July 1985 to June 1987 with a centralized registration system, and were allocated into the 5-FU+PSK group (Group P) and 5-FU alone group (Group C) by the minimization method following the random permuted blocks method. Between the two groups, the parameters of sex, age, serosal invasion (S), lymph-node metastasis (N), and the combination of S . N factor levels were distributed without significant differences. An induction treatment with MMC 6 mg/m2 was given to all patients following curative mastectomy and on the 7th post-operative day. Two weeks after surgery, Group P received alternately PSK 3 g/day for 4 week and 5-FU 150 mg/day for 4 weeks as one course, and 10 courses were given. Group C received 5-FU alone for 4 weeks using alternate rest interval for 4 weeks. Since both experimental and clinical studies suggested that alternate treatments using PSK and anticancer agents were effective, treatment in this trial alternated PSK and 5-FU. A final follow-up study will be completed in June 1992, when all patients shall have survived more than 5 years after surgery. The administration of 5-FU was completed by January. 1989, but PSK has been administered to group P. The period from 18 to 42 months after surgery was reached in all eligible patients (253) at the end of December 1988. The disease-free survival curves and overall survival curves of group P were significantly (p = 0.018 and p = 0.045,) better than those of group C.
A total of 103 patients with advanced gastric carcinoma were randomized after curative surgery to receive an alternate administration of carabzilquinone (CQ) and PSK (Krestin) or carbazilquinone alone. Each course of therapies started 1 week after the surgical operation and therapy schedules consisted of 9 courses. In each course of 6 weeks, CQ was administered on day 0, 8, and 15. In combined immunochemotherapy group, PSK was given orally in 3-divided doses of 2g/m^2/day from the day of the third CQ administration for consecutive 4 weeks. Estimated survival rate and cumulative survival curve were compared untilizing the data up to 7 years after the operation. There was no overall significant difference in survival rates between the CQ plus PSK group and the CQ alone group, but a group of patients survived significantly longer when treated with the combination of CQ and PSK. Neither in more advanced cases nor in cancers of early stages, the addition of PSK provided an additive effect. The favorable result obtained in one subgroup treated with PSK, suggests that the use of this agent in treating gastric cancers should be carefully evaluated in terms of serosal infiltration and nodal metastasis.?
Department of Zoology, The University of Hong Kong, Hong Kong, SAR, P.R. China.
Abstract
Proteins and peptide bound polysaccharides (PSP) extracted from Basidiomycetous fungi are widely used in cancer immunotherapy and recently demonstrated to induce apoptosis in cancer cells in vitro. In order to provide the molecular pharmacological mechanisms of PSP on human cancer cells, we investigated the gene expression profiles of PSP-treated apoptotic human promyelotic leukemic HL-60 cells using ResGen 40k IMAGE printed cDNA microarray. In total 378 and 111 transcripts were identified as differentially expressed in the apoptotic cells by at least a factor of 2 or 3, respectively. Our data show that PSP-induced apoptosis in HL-60 cells might be mediated by up-regulation of early transcription factors such as AP-1, EGR1, IER2 and IER5, and down-regulation of NF-kappaB transcription pathways. Other gene expression changes, including the increase of several apoptotic or anti-proliferation genes, such as GADD45A/B and TUSC2, and the decrease of a batch of phosphatase and kinase genes, may also provide further evidences in supporting the process of PSP induced apoptosis in cancer cells. Some of the well-characterized carcinogenesis-related gene transcripts such as SAT, DCT, Melan-A, uPA and cyclin E1 were also alternated by PSP in the HL-60 cells. These transcripts can be employed as markers for quality control of PSP products on functional levels. The present study provides new insight into the molecular mechanisms involved in PSP-induced apoptosis in leukemic HL-60 cells analyzed by cDNA microarray.