NK4 gene therapy combined with cisplatin inhibits tumour growth and metastasis of squamous cell carcinoma.

BACKGROUND: NK4 inhibits vascularisation in tumour tissues, thereby arresting tumour growth. However, the antitumour efficacy of individual antiangiogenic molecules expressed in vivo is not sufficiently potent to induce regression in animal models. One of the strategies to overcome this disadvantage is to use chemotherapy. MATERIALS AND METHODS: This study evaluated the efficacy of combining NK4 gene therapy with cisplatin to treat experimental squamous cell carcinomas. For gene therapy, biodegradable cationised gelatin microspheres were used for the controlled release of NK4 plasmid DNA. RESULTS: A combined regimen of antiangiogenic gene therapy and low-dose cisplatin led to a marked decrease in tumour volume and vascularity, and caused increased apoptosis compared to NK4 gene therapy alone. Moreover, combination treatment of NK4 gene therapy and low-dose cisplatin dramatically inhibited the formation of lung metastases. CONCLUSION: NK4 gene therapy combined with low-dose cisplatin may be an effective regimen for treating oral squamous cell carcinoma.

Cold shock domain protein A (CSDA) overexpression inhibits tumor growth and lymph node metastasis in a mouse model of squamous cell carcinoma.

Cancer cells metastasize by entering the lymphatic system. Regional lymph-node dissemination is the first detectable step in the metastasis of oral squamous cell carcinoma (SCC) and is highly correlated to the prognosis of the disease. Cold shock domain protein A (CSDA) is a DNA-binding protein that represses angiogenesis and lymphangiogenesis by directly binding to hypoxia response element (HRE) and serum response element (SRE). In our study we used the cell line NR-S1M, a mouse SCC model with a high rate of lymph-node metastasis. Into these cells we transfected the expression-plasmid coding for full-length mouse CSDA. Of importance, we showed that overexpression of CSDA significantly inhibits the production of VEGF-A and VEGF-C in NR-S1M cells. The overexpression of CSDA in NR-S1M cells inhibited tumor growth, inhibited regional lymph-node metastasis, and reduced the density of blood vessels and lymphatic vessels in the primary tumors in vivo. Our results support the hypothesis that VEGF-A and VEGF-C are crucial regulators of angiogenesis and lymphangiogenesis in NR-S1M cells. Therefore, they are promising targets for CSDA overexpression gene therapy to inhibit tumor growth and lymph-node metastasis in SCC.

Interleukin-12-responding asialoGM1+CD8+ central memory-type T cells as precursor cells for interferon-gamma-producing killer T cells.

While investigating CD8(+) memory T cells in unimmunized C57BL/6 mice, we found that there were unique memory-type CD8(+) T cells expressing asialoGM1 (ASGM1), CD62L and CCR7 cell surface molecules, which occupied approximately 10% of CD8(+) T cells and 35% of CD44(+) memory CD8(+) T cells. Culture of freshly isolated ASGM1(+)CD8(+) T cells with interleukin (IL)-12 plus IL-2 caused the proliferation and generation of killer T cells. Moreover, ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, produced high levels of interferon (IFN)-gamma in response to IL-12 plus IL-2. Although ASGM1(+)CD8(+) T cells showed no significant responses to IL-12 alone or IL-2 alone, pulse incubation of ASGM1(+)CD8(+) T cells with IL-12 at an earlier time (0-12 h), and subsequently with IL-2 at a later time (12-24 h), caused the same levels of proliferation, killer cell generation and IFN-gamma production as when they were incubated simultaneously with IL-12 plus IL-2 for 24 h. Thus, ASGM1(+)CD8(+) T cells appeared to respond to IL-12 directly to acquire IL-2 responsiveness and differentiate into IFN-gamma-producing killer T cells. Indeed, freshly isolated ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, expressed higher levels of IL-12R beta2 mRNA. The fact that IL-12 administration in vivo caused the generation of ASGM1(+)CD8(+) killer T cells in an IFN-gamma-dependent manner further indicated a physiological significance of ASGM1(+)CD8(+) central memory-type T cells in IL-12-induced immunoregulation for the therapy of tumors and infectious diseases.

Inverted ductal papilloma of minor salivary gland: case report with immunohistochemical study and literature review.

Inverted ductal papilloma (IDP) is a type of ductal papilloma arising in ducts of minor salivary glands. Very few cases, and no cases in Japan, have been reported. Reported herein is a case of IDP with a review of the literature. The patient was a 49-year-old man presenting with a lump in the right buccal mucosa of the premolar area of the mandible. The tumor was excised en bloc after a biopsy diagnosis of IDP. On the surface of the covering epithelium, an opening was seen to be filled with mucinous material. On cut surface the opening led to the tumor cavity. The major portion of the tumor parenchyma was made up of papillary proliferation of basaloid squamous cells. Some crypts, microcysts, and mucous cells were seen. There were no findings suggestive of a malignant tumor. The patient's postoperative course was uneventful and there has been no recurrence after 1 year's follow up. Immunohistochemical analysis of the present case supports the hypothesis that IDP originates from squamous metaplasia and proliferation of minor salivary gland duct cells.

Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma.

Double-stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21-23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi down regulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis-dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS-1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF-transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin-1 by pericytes. These data suggest that cationized-gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS-1 squamous cell carcinoma xenograft model.

Essential role of LFA-1 in activating Th2-like responses by alpha-galactosylceramide-activated NKT cells.

NKT cells produce large amounts of cytokines associated with both the Th1 (IFN-gamma) and Th2 (IL-4) responses following stimulation of their invariant Valpha14 Ag receptor. The role of adhesion molecules in the activation of NKT cells by the Valpha14 ligand alpha-galactosylceramide (alpha-GalCer) remains unclear. To address this issue, LFA-1-/- (CD11a-/-) mice were used to investigate IL-4 and IFN-gamma production by NKT cells following alpha-GalCer stimulation. Intriguingly, LFA-1-/- mice showed increased IL-4, IL-5, and IL-13 production and polarized Th2-type responses in response to alpha-GalCer in vitro and in vivo. Furthermore, the Th2-specific transcription factor GATA-3 was up-regulated in alpha-GalCer-activated NKT cells from LFA-1-/- mice. These results provide the first genetic evidence that the adhesion receptor LFA-1 has a crucial role in Th2-polarizing functions of NKT cells.