[Antitumor effect of PSK (2): effector mechanism of the antimetastatic effect in the "double grafted tumor system"].

The antimetastatic effect of PSK was analysed with the "double grafted tumor system" in which BALB/c mice received simultaneous intradermal inoculations of Meth-A in the right (10(6) cells) and left (2 X 10(5) cells) flanks and were then injected with PSK in the right tumor on day 3. PSK inhibited the growth of not only the right but also the left, non-treated tumor. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of 5 mg of PSK. On day 3, one hour after intravenous injection of cyclophosphamide, immunized spleen cells (2 X 10(7) cells/mouse) were injected into the Meth-A tumor. Adoptive transfer of PSK immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 monoclonal antibody plus complement. Spleen cells and right and left regional (axillary and inguinal) lymph node cells prepared from PSK immunized mice 7 and 14 days after tumor inoculation were examined for Thy-1, Lyt-1, Lyt-2 and asialo GM1 phenotypes by flow cytometric analysis. The number of Lyt-1 positive lymphocytes increased in the right regional lymph nodes after intratumoral administration of PSK. Immunohistochemical analyses of the right and left tumors in the "double grafted tumor system" on day 7 and day 14 were carried out by PAP (peroxidase antiperoxidase) method. Necrosis, karyoklasis and a massive accumulation of macrophages were found in the right tumor after intratumoral administration of PSK. An infiltration of macrophages and Lyt-2 positive lymphocytes was found in the left, non-treated tumor. These results suggest that intratumoral administration of PSK might induce Lyt-1 positive cells first in regional lymph nodes, then in the spleen, and subsequently induce macrophages and Lyt-2 positive cells in the left, non-treated tumor of the "double grafted tumor system", thus bringing about the regression of metastatic tumors.

[Antitumor effect of synthetic cord factor analogue, 6, 6'-Di-O-decanoyl-alpha, alpha-trehalose].

The antitumor effect of a new synthetic cord factor analogue, 6, 6'-Di-O-dacanoyl-alpha, alpha-trehalose (SS 554), was examined in vivo and in vitro. Remarkable life prolongation effects were observed after the intraperitoneal administration of SS554 at a dosage of 150 mg/kg in mice implanted with Ehrlich ascitic tumor cells. Using in vitro tests, the growth of various tumor cell lines i.e. Meth-A, EL-4, RL male-1, X5563, Raji and Jurkat were inhibited by SS554 at a 25 mcg/ml concentration. The mechanism of the direct antitumor activity of SS554 was studied using these tumor cell lines and bacterial cells. After treatment with SS554 at a 50 mcg/ml dose for 30 minutes, tumor cells and bacterial protoplasts but not intact bacterial cells were destroyed, indicating that SS554 causes the lysis of the membranes of these cells.

[Antitumor effect of PSK (1). Interferon-inducing activity and intratumoral administration].

After oral administration of 20 mg/kg of PSK, a significant level of interferon (IFN) activity was detected in the sera of BCG (1 mg/mouse)-sensitized mice. A low titer of IFN was also detected in the sera of all of 4 cancer patients after 3 g/day oral administration of PSK for 7 consecutive days. Intratumoral administration of PSK (5 mg/mouse) strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to complete regression of tumors and resistance to reinoculated tumors in the host animals. Subsequently, the antimetastatic effect of PSK was examined in a "double grafted tumor system" in which mice first received simultaneous intradermal inoculations of Meth-A in the right (10(6) cells) and left (2 X 10(5) cells) flanks and were then injected with PSK in the right tumor on day 3. PSK significantly inhibited the growth of the left, non-treated tumor. When mice received an inoculation of Meth-A (2 X 10(5) cells) in the left flank and PSK was injected subcutaneously in the right flank on day 3 (single tumor system), the growth of the tumor was not inhibited. These findings suggest that intratumoral PSK immunotherapy in one region has an effect on tumor growth in another region.

[Antitumor effect of a synthetic cord factor, 6,6'-di-O-decanoyl-alpha, alpha-trehalose (SS 554) in mice].

The antitumor effect of a synthetic cord factor (6, 6'-Di-O-decanoyl-alpha, alpha-trehalose) (SS 554) on the growth of Meth-A fibrosarcoma in BALB/c mice was examined. With regard to administration routes, only intratumoral (i.t.) injection showed a curative effect; subcutaneous (s.c.), per oral (p.o.) or intravenous (i.v.) routes has no such effect. To show the antitumor effect of known natural and synthetic cord factors, the co-presence of oily vehicles has been shown to be necessary. Accordingly, compound SS 554 examined in suspensions of sesame oil, squalane (SQA), squalene (SQE) or sesame oil and water emulsion had a curative effect with a 60% survival rate. However, no such effect was obtained with a suspension in PBS or in HCO-60 solution. In this regard, it should be noted that sequential but independent administration of SS 554 and oil was found to be equally as effective as simultaneous administration of oil with SS 554. Thus the effect of the oil should be reconsidered through an examination of the sequential appearance of effector cells. In the case of sesame oil, the amount of oil necessary was over 10%, or 0.01 mg absolutely. When the dose effect of SS 554 was examined in the presence of 10% sesame oil, doses over 1 mg exhibited a dose dependent curative effect. In tumor-bearing mice, the effect of the time of administration was also examined; the best result was obtained when intratumor injection was performed on day 3 after tumor implantation. Mice that recovered after SS 554 treatment exhibited growth inhibition and rejection of rechallenged Meth-A cells. However, this immunity was specific as it did not extend to a rechallenge with RL male-1 leukemia cells.

Antitumor effector mechanism at a distant site in the double grafted tumor system of PSK, a protein-bound polysaccharide preparation.

The antitumor effect at a distant site of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received simultaneous intradermal inoculations of Meth-A tumor in the right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with PSK in the right-flank tumor on day 3. PSK inhibited the growth of not only the right but also the left (non-treated) tumor. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of 5 mg of PSK and were injected into the Meth-A tumor on day 3. Adoptive transfer of PSK immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 monoclonal antibody plus complement. Spleen cells and right and left regional lymph node cells prepared from PSK immunized mice were examined for Thy-1, Lyt-1, Lyt-2 and asialo GM1 phenotypes. The number of Lyt-1-positive lymphocytes increased in the right regional lymph nodes after intratumoral administration of PSK. A massive accumulation of macrophages and polymorphonuclear leukocytes was found in the right tumor and an infiltration of macrophages and Lyt-2-positive lymphocytes was found in the left (non-treated) tumor by immunohistochemical analyses. These results suggest that intratumoral administration of PSK induces Lyt-1-positive cells first in regional lymph nodes, then in the spleen, and subsequently induces macrophages and Lyt-2-positive cells in the left (non-treated) tumor, thus bringing about the regression of metastatic tumors.

Antitumor effect of PSK: role of regional lymph nodes and enhancement of concomitant and sinecomitant immunity in the mouse.

PSK, a Coriolus preparation, inhibited the growth of not only the right but also the left, non-treated tumor in a double grafted tumor system. In order to examine the role of lymph nodes and the spleen in the antitumor activity of PSK, regional (axillary and inguinal) lymph nodes and spleen were resected. Since in resected mice the antitumor activity of PSK against the right and left tumors was weakened, the regional lymph nodes and the spleen probably have a very important role in the antimetastatic effect of intratumoral administration of PSK. TIL (tumor-infiltrating lymphocytes) obtained from left and right side tumors treated with PSK were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TIL from both sides clearly inhibited the growth of admixed Meth-A cells, but control TIL did not. A primary growth of Meth-A sarcoma inoculated into the right flank resulted in the generation of concomitant immunity to the growth of a second graft of the same tumor cells in the left flank. A significant inhibitory effect on the proliferation of the tumor cells inoculated secondarily was shown in mice bearing a primary right tumor that had previously been inoculated with PSK 3 times. After surgical excision of the primary tumor on day 6, daily oral administration of PSK significantly inhibited the growth of the secondary tumor inoculated on day 21, that is, PSK treatment also enhanced sinecomitant immunity. These observations suggest that presurgical intratumoral injection and postoperative oral administration of PSK are highly effective in eradicating metastatic tumors.

Antimetastatic effect of biological response modifiers in the "double grafted tumor system".

The antimetastatic effect of biological response modifiers (BRM) in a new experimental mouse model was studied. Intratumoral administration of BRMs (PSK, OK-432, interferon alpha A/D) strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete regression of tumors and resistance to reinoculated tumors. Subsequently, the antimetastatic effect of BRMs was examined in the "double grafted tumor system," in which mice first received simultaneous intradermal inoculations of Meth-A in the right (10(6) cells) and left (2 X 10(5) cells) flanks and were then injected with BRMs in the right tumor on day 3. PSK and interferon (IFN) significantly inhibited the growth of the left (non-treated) tumor. This finding suggests that intratumoral BRM immunotherapy in one region has an effect on tumor growth in another region. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of BRMs and had rejected reinoculated tumors. One hour after intravenous injection of cyclophosphamide (2 mg/mouse), immunized spleen cells (2 X 10(7) cells/mouse) were injected into the Meth-A tumor on day 3. Adoptive transfers of PSK and IFN immunized spleen cells caused the complete regression of Meth-A tumors. These results suggest that the intratumoral administration of BRMs might induce cytotoxic cells in the left non-treated tumor of the "double grafted tumor system" and bring about the regression of metastatic tumors.

The "double grafted tumor system", proposed to find effector cells in the analyses of antitumor effect of BRMs.

The antitumor effects of three biological response modifiers (BRMs; PSK, IFN alpha A/D and OK432) and two chemotherapeutics (Mitomycin C and Neocarzinostatin) in a new experimental mouse model, the "double grafted tumor system," were evaluated. BALB/c mice received simultaneous inoculations of Meth A fibrosarcoma cells on right flank (1 x 10(6) cells) and left flank (2 x 10(5) cells) on day 0, and drugs were given intratumorally into the right-flank tumor on day 3. The growth of the left-flank tumor was the real target for the evaluation of a given drug after 21 days. All tested five agents successfully cured the drug-injected right tumor with a pre-determined optimum dose. In addition, PSK, OK432, IFN alpha A/D and MMC among the five, inhibited the left-flank tumor, whereas no inhibition was observed when treated with NCS. To understand the mechanism by which the antitumor effect of the above four agents is able to influence the growth of tumor on the other side, tumor cells (2 x 10(5) cells) inoculated only into the left flank were treated with drugs given subcutaneously to the right flank (single tumor system). Among the four, MMC exhibited an effect similar to that obtained in the double tumor system, and IFN alpha A/D showed a less pronounced but still definite antitumor effect. However, PSK and OK432 failed to express anti-tumor effect in the single tumor system. These results obtained with PSK, OK432 and IFN alpha A/D suggest that the effect of the drug on the left-tumor may be mediated by certain effector cells, which are specifically induced by injection of the drug, in the right-tumor tissues. When effector cell analysis was conducted with spleen cells obtained after PSK treatment by means of intratumoral adoptive transfer into 3-day Meth A bearing recipients, these cells were shown to be Lyt-1+2(-)-T and L3T4(+)-T cell.

Antitumor effect of a synthetic cord factor, 6,6'-di-O-decanoyl-alpha,alpha-trehalose (SS554), in mice.

The antitumor effect on Meth-A fibrosarcoma in BALB/c mice of a synthetic cord factor, 6,6'-di-O-decanoyl-alpha,alpha-trehalose (designated as SS554), was examined. Only intratumoral injection had a curative effect; subcutaneous, per oral, or intravenous routes had no such effect. The co-presence of an oily vehicle has been shown to be necessary for antitumor activity of a natural cord factor. When SS554 was examined in suspensions of sesame oil, squalane, squalene or sesame oil and water emulsion, a 60% cure rate was achieved. However, no such effect was obtained with a suspension in Tween-PBS or a solution. It should also be noted that sequential but independent administrations of SS554 and oil were found to be as effective as the simultaneous administration of oil and SS554 in emulsion form. In the case of the emulsion, the amount of sesame oil necessary was over 10%, or 0.01 ml in absolute terms. Cures were obtained in a dose-dependent manner by injection of SS554 in amounts in excess of 1 mg. The effect of the time of administration was also examined; the best result was obtained when intratumoral injection was done on day 3 after tumor implantation. Mice cured by SS554 exhibited growth inhibition and rejection of rechallenged Meth-A cells. However, this immunity was specific; it did not extend to a rechallenge with RLmale-1 leukemia cells.

Disaccharide esters screened for inhibition of tumor necrosis factor-alpha release are new anti-cancer agents.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine playing a part in various pathological states. Non-toxic inhibitors of TNF-alpha release are thought to be promising agents for cancer prevention. We found that the acetone fraction of the tobacco leaf surface lipid containing glucose esters and sucrose esters inhibited both TNF-alpha release from BALB/3T3 and KATO III cells induced by okadaic acid and tumor promotion by okadaic acid on mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA). Next, we investigated the inhibition of TNF-alpha release with synthetic disaccharide esters, such as 6,6'-di-O-alkanoyl-alpha, alpha-trehaloses (6,6'-diester-trehaloses), 4,4'-di-O-alkanoyl-alpha, alpha-trehaloses (4,4'-diester-trehaloses) and 6,6'-diamino-6,6'-dideoxy-N,N'-dialkanoyl-alpha, alpha-trehaloses (6,6'-diamide-trehaloses) bearing fatty acids of various chain lengths, and n-dodecyl-beta-D-maltoside as a disaccharide monoester. 6,6'-Diester-trehaloses and 4,4'-diester-trehaloses of C8 to C12 fatty acids, 6,6'-diamide-trehaloses of C8 to C14 fatty acids, and n-dodecyl-beta-D-maltoside all inhibited TNF-alpha release in a dose-dependent manner. The IC50 values are 7.4-14.8 microM for 6,6'-diester-trehaloses (C8 to C12), 14.6-21.6 microM 4,4'-diester-trehaloses (C8 to C12), 2.9-15.0 microM for 6,6'-diamide-trehaloses (C8 to C14) and 23 microM for dodecyl-beta-D-maltoside. Both 6,6'-di-O-octanoyl-alpha, alpha-trehalose (C8, designated as SS555) and n-dodecyl-beta-D-maltoside (C12) inhibited tumor promotion by okadaic acid on mouse skin initiated with DMBA. Percentages of tumor-bearing mice in week 15 of tumor promotion were reduced from 60.0 to 13.3 with SS555, and to 46.7 with n-dodecyl-beta-D-maltoside. Moreover, SS555 inhibited TNF-alpha gene expression mediated through inhibition of AP-1 activation, but not NF-kappa B activation. This paper reports that diester-trehaloses of C8 to C12 fatty acids and mimics of disaccharide monoesters such as n-dodecyl-beta-D-maltoside appear to be potential cancer-preventive agents of a new type.