Hidehiko Kohya

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

Tumor necrosis factor‐α (TNF‐α) is a proinflammatory cytokine playing a part in various pathological states. Non‐toxic inhibitors of TNF‐α 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‐α 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‐α release with synthetic disaccharide esters, such as 6,6′‐di‐0‐alkanoyl‐α,α‐trehaloses (6,6′‐diester‐trehaloses), 4,4′‐di‐0‐alkanoyl‐α,α‐trehaloses (4,4′‐diester‐trehaloses) and 6,6′‐diamino‐6,6′‐dideoxy‐N, N′‐dial‐kanoyl‐α,α‐trehaloses (6,6′‐diamide‐trehaloses) bearing fatty acids of various chain lengths, and n‐ dodecyl‐β‐D‐maltoside as a disaccharide monoester. 6,6′‐Diester‐trehaloses and 4,4′‐diester‐treha‐loses of C8 to C14 fatty acids, 6,6′‐diamide‐trehaloses of C8 to C14 fatty acids, and n‐dodecyl‐β‐D‐maltoside all inhibited TNF‐α release in a dose‐dependent manner. The IC50 values are 7.4‐14.8 μM for 6,6′‐diester‐trehaloses (C8 to C12), 14.6‐21.6 μM 4,4′‐diester‐trehaloses (C8 to C12), 2.9‐15.0 μM for 6,6′‐diamide‐trehaloses (C8 to C14) and 23 μM for dodecyl‐β‐D‐maltoside. Both 6,6′‐di‐O‐octanoyl‐α,α‐trehalose (C8, designated as SS555) and n‐dodecyl‐β‐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‐β‐D‐maltoside. Moreover, SS555 inhibited TNF‐α gene expression mediated through inhibition of AP‐1 activation, but not NF‐αB activation. This paper reports that diester‐trehaloses of C8 to C12 fatty acids and mimics of disaccharide monoesters such as n‐dodecyl‐β‐D‐maltoside appear to be potential cancer‐preventive agents of a new type.

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 (106 cells) and left (2 × 105 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.

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, IFNα 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 × 106 cells) and left flank (2 × 105 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, IFNα 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 × 105 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 IFNα 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 IFNαA/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.