Juichiro Nakayama

New nortriterpenoid isolated from anti-rheumatoid arthritic plant, Tripterygium wilfordii, modulates tumor growth and neovascularization

Preparations of Tripterygium wilfordii, “Thunder God vine”, have been used in China to treat rheumatoid arthritis. Rheumatoid arthritis, as well as solid tumors, is closely associated with neovascularization. Antiarthritic drugs therefore may modulate tumor growth as well as neovascularization. We found that a compound purified from T. wilfordii, the nortriterpenoid, demethylzeylasteral (TZ‐93), inhibited the proliferation of vascular endothelial cells approximately 30 times more effectively than it did for the proliferation of human tumor cells. In in vitro assays using bovine aortic endothelial cells, TZ‐93 at non‐toxic doses inhibited cell migration, expression of urokinase‐type plasminogen activator (uPA) mRNA and uPA activity. Exogenous addition of uPA restored the inhibitory effect of TZ‐93 on cell migration. In dorsal air‐sac assays in BALB/c mice, the oral administration of 3 mg/kg/day TZ‐93 for 5 days partially inhibited, and 30 mg/kg/day almost completely abrogated, the development of capillary networks induced by human hepatoblastoma cells. Similarly, 0.3 mg/kg/day TZ‐93 partially inhibited, and 3 or 30 mg/kg/day almost completely blocked, the growth of mouse B16‐F10 melanoma cells in a tumor implantation assay. The highest dose of TZ‐93 significantly reduced the growth of well‐vascularized tumors with volumes of more than 500 mm3. TZ‐93 treatment of tumor‐bearing mice significantly decreased the density of microvessels in the tumors. We conclude that TZ‐93 may be useful in treating highly vascularized and metastatic tumors as well as other angiogenic diseases. Int. J. Cancer 72:657–663, 1997.

ALOPECIA UNIVERSALIS TREATED WITH ORAL CYCLOSPORINE A AND PREDNISOLONE: IMMUNOLOGIC STUDIES

Alopecia universalis is a refractory condition. Although the cause of this disease is unknown, immunologic abnormalities have recently been suspected. Thus, we treated six cases of refractory alopecia universalis with immunotherapy. Oral administration of cyclosporine A (2.5 mg/kg) and prednisolone (5 mg/day) resulted in marked symptomatic improvement. Cyclosporine A did not produce any side effects because the administered dosage was relatively low. At present, more than 6 months after the cessation of treatment, recurrence of alopecia has not been seen. Oral administration of low‐dose cyclosporine A and prednisolone is considered to be an effective treatment for this disease. Immunologic examination of peripheral blood demonstrated improvement of immunologic function. In particular, cos‐positive T cells, NK cells, and C3, which had been reduced, were increased. A reduction in active CD4 cells, eosinophils, and circulating immune complexes was observed. Histology with fluorescent antibodies showed T‐cell infiltration around the hair matrixes. This phenomenon was no longer observed after treatment. These improvements in immunologic function were seen in parallel with the resolution of the clinical symptoms, indicating that immunologic abnormalities are related to this disease.

Malignant Granular Cell Tumor

A malignant granular cell tumor (MGCT) appeared on the subungual tissue of the right index finger of a 51‐year‐old woman. Two years after resection of the tumor, it recurred, and the finger finally had to be amputated. Six months later, she noticed multiple cutaneous nodules on her trunk. Despite chemotherapy and X‐ray irradiation, the patient died 18 months after the second operation. Histology of the specimen revealed a proliferation of both polygonal and spindle‐shaped cells with large hyperchromatic nuclei and an eosinophilic granular cytoplasm. Peripheral nerves were encompassed by the tumor cells. Immunohistochemically, the tumor cells were positive for S‐100 protein and Leu 7 (myelin‐associated glycoprotein). These findings support the hypothesis that MGCT cells are of Schwann cell origin.

Distribution of Epithelial Membrane Antigen in Eccrine Poroma

Using immunohistochemical methods, we investigated the distribution of epithelial membrane antigen (EMA) on the normal eccrine gland, eccrine poroma and hidroacanthoma simplex. Granular membrane-associated reaction of EMA was detected on the outer cells of both the intraepidermal and the upper portion of intradermal eccrine ducts, as well as on the luminal surfaces and intercellular canaliculi of eccrine glands. Clear immunolabeling was also present in the tumor cells of eccrine poroma and hidroacanthoma simplex. Thus, it is suggested that the constituent cells of these tumors originate from the outer cells of the intraepidermal and/or the upper portion of the intradermal eccrine ducts. There was no immunolabeling for EMA on the tumor cells of seborrheic keratosis and basal cell carcinoma. Immunohistochemical staining for EMA is a useful tool for the diagnosis of skin appendage tumors.

A New Apparatus for Hair Regrowth in Male-pattern Baldness

A newly devised apparatus (Scalp‐Tension‐Relaxer, STR) can efficiently promote hair regrowth in patients with male‐pattern baldness. When this apparatus is applied, the scalp is pushed up to relieve tension on the vertex. The efficacy rate of hair regrowth in alopecia patients was 40%. An investigation into the basis for the hair regrowth caused by this apparatus was directed toward the changes in hemodynamics and skin temperature of the scalp. During and after use of this apparatus, subjects exhibited an increase both in the cutaneous blood flow rate (as determined by laser Doppler flowmetry) and in the cutaneous temperature (as determined by thermography).

Immunohistochemical Characterization of Cellular Infiltrates in Epidermal Tumors Induced by Two-stage and Complete Chemical Carcinogenesis in Mouse Skin

We investigated the population and pattern of the infiltrated cells in both benign and malignant epidermal tumors which were induced chemically with benzo(a)pyrene (B(a)P) in murine skin. In benign papillomas, which were evolved by a two stage carcinogenesis regimen, a slight to mild inflammatory infiltration around the tumors was observed, and cells infiltrating into the tumor nests were rarely seen. In carcinomas, which were produced by a complete carcinogenesis regimen, a dense inflammatory infiltration was observed around the tumor nests. The infiltrated cells were characterized as T‐lymphocytes, macrophages, and neutrophils. Natural killer (NK) cells were found around and in the tumor nests, but their number was small. Both T‐lymphocytes and macrophages were found to invade the tumor nests in squamous cell carcinoma whose duration was more than four weeks. This experimental carcinogenesis animal model allows the detailed quantitative and functional analysis of the infiltration of immunocompetent cells into epidermal tumors.

Responses of B16 melanoma cell lines, F1 and F10, to hyperthermia, lymphokine‐activated killer cells and a combination of both in vitro

The cytolytic and/or cytostatic effects of hyperthermia, lymphokine‐activated killer cells (LAK cells) and the combination of both were assayed using F1 and F10 B16 melanoma cell lines. F10 cells with a high metastatic potential showed a greater sensitivity to hyperthermia than F1 cells which have low metastatic potential. The F10 cells were lysed to a lesser extent by LAK cells than the F1‐B16 cells. When the cell lines were subjected to hyperthermia at 43°C for 3 h and then interacted with LAK cells, the maximum cytolysis reached almost 100%. When the interaction with LAK cells was followed by hyperthermia at 43°C, the total release of 51Cr from the cell lines was 75–85%. The extent of 51Cr release from the B16 melanoma cell lines was inversely correlated with the survival rate as calculated by the plating efficiency of the incubated cells. The survival rate of mice intravenously injected with B16‐F10 cells and subjected to hyperthermia at 41°C for 3 h in vitro increased compared to that of controls. This was further increased by the simultaneous administration of LAK cells.

A combined therapeutic modality with hyperthermia and locally administered rIFN-β inhibited the growth of B16 melanoma in association with the modulation of cellular infiltrates

Murine B16-F10 melanoma was treated with local microwave hyperthermia, local injection of murine recombinant interferon-beta (rIFN-beta) or a combination of both in order to investigate the augmentation of anti-proliferative effects with this combination treatment. Concerning the local modulation of immunological reactions of the host, local hyperthermia at 43 degrees C for 15 min on murine melanoma caused remarkable infiltration of natural killer cells and local injection of rIFN-beta led to considerable infiltration of T cells. When these two modalities were combined, the infiltration of NK cells completely disappeared and, instead, remarkable augmentation of T cell infiltration occurred. Synergistic suppressive effects on melanoma growth with occasional scar formation were seen with this combined modality. These results indicate that local hyperthermia with a combination of rIFN-beta modulates local immune reactions of the host, and probably this immune reaction is partly involved in the course of the suppression of tumor growth.

Differential expression of smooth muscle α-like actin between benign and malignant human pigment tissues

When examining proteins in human pigment tissues, we found that a third actin-like protein, in addition to beta- and gamma-actin was more frequent and in a larger amount in benign tissues such as blue nevus and nevus pigmentosus, than in malignant melanoma. This third actin-like protein was immunologically stained with monoclonal antibodies reacting with several actin species and specific for smooth muscle alpha-actin. We propose that this third actin-like protein is probably smooth muscle alpha-actin and that different expressions of this third actin may possibly serve as a sensitive biochemical marker for the diagnosis of human malignant melanoma.

Hyperthermic Isolated Limb Perfusion with Intra‐Arterial Administration of Carboplatin and/or Interferon‐β for the Treatment of Malignant Melanoma of the Leg

Our experiences of hyperthermic isolated limb perfusion with administration of carboplatin, interferon‐β or a combination of both are reported. Administration of high doses of these reagents was well tolerated by patients with melanoma without severe complications after the treatment. A total of 8 patients underwent this therapeutic modality. Remarkable clinical improvement was seen in the first patient, who was in Stage III at the time of the perfusion. Histopathological findings indicated severe damage to the melanoma cells after the operation. Prophylactic hyperthermic perfusion was performed in 6 other patients with Stage II–III melanoma of the lower limb. None of them have shown any signs of recurrence 1–10 months later. The activities of natural killer cells or T lymphocytes appeared to be increased when the perfusion was carried out with concomitant administration of both carboplatin and interferon‐β. These results suggest that hyperthermic isolated limb perfusion with carboplatin and/or interferon β administration is effective in patients with advanced stage melanoma.