Kuniaki Tanaka

Antitumor effect induced by a hot water extract of Chlorella vulgaris (CE): Resistance to meth-A tumor growth mediated by CE-induced polymorphonuclear leukocytes

SummaryWhen a hot water extract of Chlorella vulgaris (CE) was injected into the peritoneal cavity of BALB/c mice inoculated with syngeneic Meth-A tumor cells, the survival times were strikingly prolonged. Furthermore, peritoneal exudate cells (PEC) rich in polymorphonuclear cells (PMN) obtained from normal mice 24 h after CE injection exhibited an antitumor effect in a Winn-type assay using normal recipients. Such an activity of PEC remained almost intact after T cell or macrophage depletion. However, such PEC did not express an antitumor effect in a Winn-type assay using irradiated recipients. It was suggested that CE-induced PEC, presumably PMN, expressed an antitumor effect in cooperation with a host- or recipient-derived element(s) sensitive to irradiation. The anti-tumor mechanism of CE may be different from that of OK-432, one of the biological response modifiers.

Augmentation of antitumor resistance by a strain of unicellular green algae, Chlorella vulgaris

SummaryGrowth of Meth-A tumor in CDF1 mice was inhibited significantly by injection of a hot water extract of a strain of Chlorella vulgaris (CE) into the tumor or into the subcutaneous tissue near the tumor. The augmentation of resistance by CE may require the participation of T cells and macrophages, since it was abolished or reduced in athymic nude mice or mice treated with carrageenan, a macrophage blocker. Mice treated with CE exhibited antigen-specific augmented resistance against rechallenge with tumor. Moreover, the antitumor effect of CE was comparable with that of Corynebacterium parvum, but its mechanism of effect might be different.

A novel glycoprotein obtained from Chlorella vulgaris strain CK22 shows antimetastatic immunopotentiation

Abstract A glycoprotein extract (CVS), derived from the unicellular green alga Chlorella vulgaris, strain CK22, exhibited a pronounced antitumor effect against both spontaneous and experimentally induced metastasis in mice. Inhibition of tumor metastasis was enhanced when intratumor administration of CVS was followed by s.c. injection of CVS. Anti-metastatic immunopotentiation was observed in euthymic mice, but not in athymic nude mice. The antitumor activity of CVS was reflected in antigen-specific, T-cell-mediated immunity. Both CD4 and CD8 T cells contributed to the antimetastatic effects, as shown by in vivo depletion experiments with anti-T-cell subset antibodies. Furthermore, CVS caused the recruitment of T cells to the regional lymph nodes and their proliferation in these organs. The CD4-positive population, following CVS injection at the time of tumor rechallenge, displayed a pronounced increase in the proportion of T cells that were CD18 bright, CD44 bright, CD25+, CD54+, CD69+ or CD71+ in the lymph nodes. Thus, CVS induces T cell activation in peripheral lymph nodes in tumor-bearing mice. We conclude that CVS augments antimetastatic immunity through T cell activation in lymphoid organs and enhances recruitment of these cells to the tumor sites. Presurgical treatment with CVS might prevent metastasis or tumor progression.

Protective effect of an acidic glycoprotein obtained from culture of Chlorella vulgaris against myelosuppression by 5-fluorouracil

Abstract An acidic glycoprotein prepared from a culture of Chlorella vulgaris (CVS) was examined for its protective effect on 5-fluorouracil(5FU)-induced myelosuppression and indigenous infection in mice. Subcutaneous administration of CVS greatly reduced the mortality of non-tumor-bearing mice given a high dose of 5FU, and could increase the LD50 value of 5FU for these mice. After 5FU treatment, indigenous infection developed probably as a result of the impairment of the host defense system. CVS reduced the incidence of indigenous infections and this effect was attributable to the acceleration of recovery from 5FU-induced myelosuppression. Early recovery of hematopoietic stem cells, or cells responding to interleukin-3 or granulocyte/macrophage-colony-stimulating factor, was especially observed in the bone marrow of CVS-treated mice on days 4 – 9 after the injection of 5FU. When tumor-bearing mice were given CVS during treatment with 5FU, CVS prolonged the survival of mice without affecting the antitumor activity of 5FU. In addition, CVS was itself shown to exert an antitumor effect. These results suggested that CVS may be beneficial for the alleviation of side-effects in cancer chemotherapy without affecting the antitumor activity of the chemotherapeutic agent.

Toll-like receptor 2 is at least partly involved in the antitumor activity of glycoprotein from Chlorella vulgaris.

Toll-like receptors (TLR) are involved in innate immunity by recognizing various bacterial components. We have previously reported that an active substance of ARS-2 purified from the culture medium of Chlorella vulgaris was a glycoprotein with a molecular weight of 63,100 amu and that this glycoprotein expressed antitumor activity, with the protein moiety in ARS-2 being necessary for this antitumor activity. Here, we show that ARS-2 stimulated spleen-adherent cells from C3H/HeJ lacking functional TLR4 to produce interleukin-12 (IL-12) p40, whereas such cytokine production was significantly impaired in ARS-2-stimulated spleen-adherent cells from TLR2 knockout mice. The overexpression of mouse TLR2 (mTLR2) and mouse CD14 (mCD14) conferred the ARS-2 inducibility of nuclear factor-kappaB activation to human HEK 293 cells. These results suggest that TLR2 signaling is at least partly involved in the antitumor activity of the water-soluble antitumor glycoprotein from C. vulgaris.

Enhanced resistance againstEscherichia coli infection by subcutaneous administration of the hot-water extract ofChlorella vulgaris in cyclophosphamide-treated mice

SummaryThe effects ofChlorella vulgaris extract (CVE-A) on the recovery of leukocyte number and the augmentation of resistance to bacterial infection were examined in CDF1 mice made neutropenic by cyclophosphamide (CY). They were treated intraperitoneally with CY (150 mg/kg) on day 0, and were given CVE-A (50 mg/kg) subcutaneously (s. c.) every other day from day 1 to day 13 after CY treatment. CVE-A accelerated the recovery of polymorphonuclear leukocytes (PMN) in the peripheral blood in CY-treated mice. The number of granulocyte/monocyte-progenitor cells (CFU-GM) in the spleen increased rapidly and highly after the administration of CVE-A in CY-treated mice, in contrast to the absence of change due to CVE-A in the number of bone marrow cells in CY-treated mice. Administration of CVE-A in CY-treated mice enhanced the accumulation of PMN in the inflammatory site and the activity of the accumulated leukocyte cells in luminol-dependent chemiluminescence. The mice became highly susceptible to an intraperitoneal infection withE.coli on day 4 after CY treatment, whereas the mice given CVE-A showed an enhanced resistance againstE.coli infection, irrespective of the timing of challenge. The bacterial number in CY-treated mice increased explosively after inoculation, resulting in death within 24 h. A progressive elimination of bacteria was observed from 6 h in the peritoneal cavity, spleen and liver of CY-treated mice given CVE-A s.c. These results indicate that CVE-A can be used as a potent stimulant of nonspecific resistance to infection in neutropenic mice.

Augmentation of the resistance against Escherichia coli by oral administration of a hot water extract of Chlorella vulgaris in rats.

In previous studies, we demonstrated that a hot water extract of Chlorella vulgaris (CVE) augmented the resistance against an intraperitoneal infection with Escherichia coli by its intraperitoneal, intravenous or subcutaneous administration. The augmented resistance appeared to be attributable to the enhanced activity of polymorphonuclear leukocytes (PMN). In this study, the effect of oral administration of CVE against Escherichia coli infection was examined. Male Fisher rats (F344/DuCrj) were administered 1000 mg/kg of CVE orally for 14 days and challenged with 2.7 x 10(8) Escherichia coli intraperitoneally. The numbers of living bacteria in the peritoneal cavity, blood, spleen and liver at 1, 6, and 24 h after the inoculation were counted. The bacterial numbers increased during 1-6 h and reached the peak at 6 h in both control and CVE-administered groups. The bacterial numbers decreased to an undetectable level at 24 h in both groups. In a CVE-administered group, the numbers of viable bacteria in each organ were remarkably lower than those in a control group in all organs so far tested. Whereas, the leukocyte numbers, especially PMN numbers, in the peritoneal cavity and peripheral blood maintained higher levels in the CVE-administered group at 6 h after E. coli inoculation. Chemiluminescent responses of peritoneal exudate cells induced by casein or E. coli were higher in a CVE-administered group. These results form the basis for the judgment that the degree of effectiveness of bacteria clearance from the peritoneal cavity shown by oral CVE administration may be strong enough to warrant developing this material as a new type of biological response modifier.

A new type of biological response modifier from Chlorella vulgaris which needs protein moiety to show an antitumour activity

An immunopotentiator obtained from Chlorella vulgaris strain CK22, showed antitumour effects against various lines of syngeneic tumours, especially by intratumour administration. The immunopotentiator exhibited far greater antitumour activity against a rechallenged tumour than against the primary‐inoculated tumour in Meth A and BALB/c or CDF1 mouse systems. The antitumour effect was at least comparable to that of a streptococcal preparation, OK‐432, which has been widely used for clinical immunotherapy.

Thymus cell migration in a prostaglandin-mediated system.

Prostaglandin (PG)-mediated T cell traffic and the nature of these emigrant T cells were analyzed by using a fluorescent activated cell sorter. Administration of indomethacin (INDO), an inhibitor of PG synthesis, increased the number of splenic T cells in normal mice but not in adult-thymectomized mice. An increase in thymus cell migrants in peripheral blood lymphocyte and splenic cell populations of mice pretreated with INDO were detected, using the method of in situ labelling of thymocytes with fluorescein diacetate. These results indicate that the increase in the T cell population in the spleen by INDO treatment results from the increase in thymus cell migration to the spleen. Such recent emigrants in the spleen were thought to have been derived from the thymus cortex, judging from the response to phytohemagglutinin and intracellular terminal deoxynucleotidyl transferase activity; however, they expressed a Thy-1 level similar to that found on peripheral T cells. These results suggested that a cortical thymocyte population was recruited from the thymus to the spleen by a PG-mediated system, but its Thy-1 level rapidly changed to that found on the peripheral T cell population.

Immunopotentiating effects of a glycoprotein from chlorella vulgaris strain CK and its characteristics

Abstract Chlorella vulgaris strain CK, a unicellular greenalga, has been used as a health food for the past 30 years in Japan and in other countries. Oral administration of C. vulgaris results in several pharmacological effects, including augmenting host defenses in animal models and in human experiments. The oral administration of C. vulgaris showed clearprophylactic effects in stress-induced peptic ulcer models, presumably through the “immune-brain-gut” axis, and it also suppressed a Meth A tumor growth in an antigen-specific manner. C. vulgaris in active form, known as CVS, was purified from the culture supernatant of C. vulgaris and found to be a glycoprotein with a molecular weight of 63,100 amu. CVS contains 67% carbohydrate with a β-l,6-D-galactopyranose backbone and 35% protein. A protein moiety is essential for CVS to exhibit immunopotentiating activity, and 15-mer of the partial amino acid sequence at the NH2-terminus has been determined. CVS exhibited a pronounced antitumor effect against both spontaneous and experimentally induced metastasis by intratumor (i.t.) injection. Prophylactic effects of CVS were observed on 5-fluorouracil-induced myelosuppression and indigenous infection by subcutaneous injections. From these results, it became evident that CVS augments antimetastatic immunity through T cell activation in lymphoid organs and accelerates recruitment of these cells to the tumorsites. Presurgical treatment with CVS might prevent metastasis and/or the progression of residual tumors. CVS may also be beneficial for the alleviation of adverse effects of cancer chemotherapy, causing an early recovery of hematopoietic stem cells without affecting the antitumor activity of chemo-therapeutic agents.