Reovirus oncolysis in human head and neck squamous carcinoma cells

OBJECTIVE: To determine whether reovirus, a double-standed RNA virus is effective on the growth of a human head and neck squamous cell carcinoma cell line. DESIGNS: In vitro cell proliferation assay, KB cells, a human oral floor squamous cell carcinoma cell line, were treated with reovirus and the number of cells was quantitated by an assay, using trypan blue staining. In vivo tumor growth assay, KB cells were injected subcutaneously into athymic nude mice, which were given an intratumoral injection of reovirus to a maximum four times in every week. The tumor size was measured once a week. Simultaneously, apoptosis and necrosis of KB cells were investigated, using technique of immunohistochemistry. RESULTS: In vitro, the multiplication of the KB cell was inhibited depending on the concentration of reovirus. In vivo, athymic nude mice bearing KB tumors were injected with the virus intratumorally, and the tumor growth was suppressed proportionally depending on the injection time of reovirus. Necrosis was recognized extensively in the pathological specimen. On the other hand, apoptosis-inducing effect was not obvious in these specimen. CONCLUSIONS: Reovirus suppressed tumor growth of KB cells in vivo as well as in vitro. The possibility that reovirus could become the means of treatment for head and neck carcinoma, was suggested with further work.     

Circulating p53 Antibody in Patients with Colorectal Cancer

The presence of serum anti-p53 antibody has been reported to be associated with survival of patients with breast cancer, ovarian cancer, and hepatocellular carcinoma. To clarify prognostic significance of p53 antibody in colorectal cancer, serum p53 antibody was measured in patients with colorectal cancer. The 89 patients included 71 with colorectal cancer and 18 with colon polyp. An enzyme-linked immunosorbent assay was used to detect p53 antibodies in serum. Clinicopathological parameters such as age, sex, degree of differentiation of cancer, location of tumor, liver metastasis, stage classification, Dukes classification, CEA, CA19-9, and immunostaining of p53 and anti-p53 antibody were evaluated as prognostic factors of colorectal cancer. p53 antibody was positive in 18 of 71 (25%) with colorectal cancer, whereas it was positive in only 1 of 18 (6%) with colon polyp. The patients with p53 antibody had higher CEA and CA19-9 levels, higher positive rates of p53 protein expression in cancer cells, and higher liver metastasis rates. The p53 antibody positivity at stage classification I–IIIb/Dukes classification A–C was significantly lower than that at stage classification IV/Dukes classification D. Overall survival in colorectal cancer patients with p53 antibody was significantly shorter than in those without p53 antibody. A Cox regression analysis showed that liver metastasis, stage classification, Dukes classification, CA19-9, and p53 antibody were significant prognostic factors in colorectal cancer. Serum anti-p53 antibody could serve as one of the prognostic factors in patients with colorectal cancer.     

Establishment of PDX‐derived salivary adenoid cystic carcinoma cell lines using organoid culture method

To generate a reliable preclinical model system exhibiting the molecular features of salivary adenoid cystic carcinoma (ACC) whose biology is still unclear due to the paucity of stable cell cultures. To develop new in vitro and in vivo models of ACC, the techniques of organoid culture and patient‐derived tumor xenograft (PDX), which have attracted attention in other malignancies in recent years, were applied. Tumor specimens from surgically resected salivary ACC were proceeded for the preparation of PDX and organoid culture. The orthotopic transplantation of patient‐derived or PDX‐derived organoids was demonstrated into submandibular glands of NSG mice and those histology was evaluated. PDX‐derived organoid cells were evaluated for the presence of MYB‐mediated fusion genes and proceeded for in vitro drug sensitivity assay. Human ACC‐derived organoids were successfully generated in three‐dimensional culture and confirmed the ability of these cells to form tumors by orthotopic injection. Short‐term organoid cell cultures from two individual ACC PDX tumors were also established that maintain the characteristic MYBL1 translocation and histological features of the original parent and PDX tumors. Finally, the establishment of drug sensitivity tests on these short‐term cultured cells was confirmed using three different agents. This is the first to report an approach for the generation of human ACC‐derived organoids as in vitro and in vivo cancer models, providing insights into understanding of the ACC biology and creating personalized therapy design for patients with ACC.     

STUDIES ON THE PATHOGENESIS OF STAPHYLOCOCCAL INFECTION : I. THE EFFECT OF REPEATED SKIN INFECTIONS

The influence of repeated staphylococcal infection of rabbit skin upon the characteristics of the experimentally induced lesion was studied. It was found that the repeated infection was associated with the development of delayed hypersensitivity unaccompanied by the appearance of demonstrable serum antibody. The delayed hypersensitivity to the staphylococcus resulted in an increased infectivity of the organism in skin of the sensitized animal, characterized by intensification of the lesions seen with large bacterial inocula and the induction of abscesses with inocula incapable of producing any lesion in normal rabbit skin. Similarly, the severity of experimentally induced pyoarthrosis was greater in sensitized than in normal rabbits. Induction of delayed hypersensitivity by vaccination of rabbits with washed heat-killed staphylococci resulted in the same increased severity of the infection and an increase in infectivity of the microorganism. In contrast to the observations of cutaneous and joint infection, the sensitized animals appeared to be less susceptible to severe infection of the anterior chamber of the eye. The role of immunity and hypersensitivity in staphylococcal infection is discussed and the possibility that non-specific inflammation may influence staphylococcal infection in the same way as specific hypersensitivity is indicated. Studies to further elucidate this are presented in the following pages.     

STUDIES ON THE PATHOGENESIS OF STAPHYLOCOCCAL INFECTION : III. THE EFFECT OF TISSUE NECROSIS AND ANTITOXIC IMMUNITY

Necrosis of rabbit skin produced by thermal injury was found to result in a striking increase in local infectivity of staphylococci that were coagulase-positive and hemolytic, but no local increase in the infectivity of non-pathogenic staphylococci. Infection produced in necrotic burns extended beyond the area of burn and was characterized by hemorrhage, edema, and necrosis of contiguous normal skin. Such infections, however, never resulted in bacteriemia or metastatic abscesses, and there was no effect of the necrotic burn upon the infectivity of staphylococci injected into normal skin of the burned animal. Recovery of rabbits from severe burn infections was associated with the development of high titers of serum antibody to the alpha hemolysin or dermonecrotoxin of the staphylococcus. Thirty to 100 days after the initial burn infection, it was found that rabbits could no longer be infected in a necrotic burn, although infection induced in normal skin of these resistant animals was no different from that in normal rabbits. Immunity to infection by pathogenic staphylococci in necrotic burns could be induced by vaccination with potent alpha hemolysin toxoid, and this immunity was passively transferable with rabbit antiserum. No strain specificity was detected for this immunity in that immunization with toxoid prepared from bacteriophage type 52/42B/80/81 staphylococci protected animals against infection in a necrotic burn by other typable and non-typable staphylococci. Histopathological study of infected necrotic burns in normal rabbits showed extensive necrosis, hemorrhage, edema, and many masses of bacteria but leucocytic infiltration was observed only at the margin of the infection. In contrast, the infected necrotic burns in animals immunized with alpha hemolysin toxoid showed few bacteria and marked leucocytic infiltration throughout the burn. These experiments have, therefore, demonstrated a significant immunity to infection by pathogenic staphylococci in necrotic tissue but not in normal skin, associated with serum antibody to the alpha hemolysin or dermonecrotoxin of the bacteria. The implications of these findings are discussed.     

Concurrent chemoradiotherapy for T4 patients with hypopharyngeal and laryngeal squamous cell carcinomas

OBJECTIVES: Concurrent chemoradiotherapy (CCR) was given for the previously untreated T4 hypopharyngeal and laryngeal squamous cell carcinoma patients and the response and survival rates were evaluated. PATIENTS AND METHODS: A total of 23 patients, namely, 15 for hypopharynx and 8 for larynx were eligible. These patients were given cisplatin and 5-fluorouracil based chemotherapeutic regimens with conventional radiotherapy for a total dose of 66.6-70.2 Gy. RESULTS: Ten out of the 15 hypopharyngeal carcinoma patients and 4 out of the 8 laryngeal carcinoma patients showed a complete response at the primary sites. The 5-year disease-specific survival rate was 59.4% in all the patients, 51.9% in the hypopharyngeal carcinoma patients, and 71.0% in the laryngeal patients. Seven out of the 12 resectable hypopharyngeal carcinoma patients and 4 out of 8 laryngeal carcinoma patients were able to do without total laryngectomy. CONCLUSIONS: Based on these results, the survival rate in the hypopharyngeal and laryngeal T4 carcinoma patients treated by CCR seems to be satisfactory and the possibility of organ preservation for the advanced patients is indicated.     

Treatment results and prognostic factors for advanced squamous cell carcinoma of the head and neck treated with concurrent chemoradiotherapy

Objective

To review our experience in the treatment of concurrent chemoradiotherapy (CCR) for patients with advanced squamous cell carcinoma of the head and neck (SCCHN) and to evaluate the different factors affecting survival and primary organ preservation.

Methods

We reviewed the records of 101 patients with SCCHN treated with CCR between February 1998 and April 2004. Of 101 patients, 76 were treated with a cisplatin, 5-fluorouracil, methotrexate, and leucovorin (PFML) regimen and 25 were treated with a carboplatin and uracil-tegafur (CBDCA-UFT) regimen. Overall survival (OS), disease-specific survival (DSS) and DSS with primary organ preservation were estimated using Kaplan-Meier methods. The log-rank test and Cox proportional hazards regression were employed to identify significant prognostic factors for OS, DSS, and DSS with primary organ preservation.

Results

The 5-year OS and DSS for all patients were 51.6 and 67.4%, respectively. On multivariate analysis, resectability of the tumor and degree of histological differentiation were significant predictors of survival for patients undergoing CCR; T stage and differentiation were significant prognostic factors for primary organ preservation.

Conclusion

In the treatment of CCR for advanced SCCHN, the survival rate of the patients with resectable tumors was excellent and significantly greater compared with the patients with unresectable tumors. T1 to T3 disease in patients with advanced resectable SCCHN is a good predictor of organ preservation. CCR may improve not only primary organ preservation (local control) but also survival in patients with poorly differentiated tumors.     

The efficacy and safety of concurrent chemoradiotherapy for maxillary sinus squamous cell carcinoma patients

Objective

Combined treatment modality, e.g., definitive surgery followed by radiotherapy (RT) and definitive RT with concurrent chemotherapy, has been applied for advanced maxillary sinus squamous cell carcinoma (MSSCC) patients to obtain a better survival with organ preservation in Japan.

Methods

The outcome of 40 patients with MSSCC between 1991 and 2007 in our institute was analyzed retrospectively. There were 36 males and 4 females, the average age being 59.5 years (ranging from 34 to 81 years). The median follow-up time was 66.1 months. All the patients had received a combined treatment consisting of definitive surgery, RT, and intra-arterial or systemic chemotherapy. The chemotherapeutic regimen was different depending on the performance status and/or complications of the patients. Since 1998, concurrent chemoradiotherapy with cisplatin, 5-fluorouracil, methotrexate and leucovorin regimen (CCRT–PFML) instead of neo-adjuvant chemotherapy has been applied.

Results

The overall 5-year survival rate was 59.2%, the 5-year disease-specific survival rate was 71.7%, and the 5-year organ preservation survival rate was 42.4%. In the group receiving CCRT–PFML, the overall 5-year survival rate was 60.0%, the 5-year disease-specific survival rate was 76.0%, and the 5-year organ preservation survival rate was 60.3%.

Conclusion

CCRT–PFML for advanced MSSCC patients is feasible to preserve the organs without reducing the survival rate.     

Inhibition of beta-adrenergic signaling by intracellular AMP is independent of cell-surface adenosine receptors in rat cardiac cells

We report a novel action of intracellular adenosine monophosphate (AMP) to inhibit beta-adrenergic signaling in isolated rat ventricular myocytes. Extracellular application of adenosine or AMP suppressed isoproterenol (Iso)-induced prolongation of action potential duration (APD). This effect was completely abolished by an A(1)-receptor antagonist, DPCPX. Intracellular application of AMP, but not adenosine, attenuated Iso-induced APD prolongation. Iso-induced increases in the L-type Ca(2+) current (I(Ca,L)) were also inhibited by intracellular AMP. These inhibitory effects were not affected by either DPCPX or glibenclamide. In vitro, AMP directly inhibited PKA activity via binding to its regulatory subunit. These results suggest that intracellular AMP attenuates beta-adrenergic signaling by directly inhibiting PKA activity, independently of A(1)-purinergic receptor.     

Regulation of hepatic oval cell proliferation by adenoviral mediated hepatocyte growth factor gene transfer and signal transduction inhibitors

BACKGROUND/AIMS: To rescue patients with severe liver injury, it is critical to develop the efficient regulatory system of hepatic stem cell proliferation in vitro. Our aims are to examine whether combination of adenovirus-mediated hepatocyte growth factor (HGF) gene transfer with signal transduction inhibitors can regulate cell proliferation of oval cells. METHODOLOGY: We examined the effects of treatment with adenoviral mediated HGF gene transfer and signal transduction inhibitors including LY294002, rapamycin and U0126 on proliferation OC/CDE22 hepatic oval cells and expression of signal transduction molecules. RESULTS: Infection with pAxCAHGF expanded the cells by 8-fold at 2 days, by 18-fold at 3 days and by 55-fold at 4 days. The addition of inhibitors inhibited pAxCAHGF-induced cell proliferation by LY294002 or rapamycin (P < 0.01, each). U0126 also inhibited growth of hepatic oval cells (P < 0.01). pAxCAHGF treatment induced phosphorylation of AKT. Treatment with rapamycin resulted in enhanced phosphorylation of AKT, and phosphorylation of AKT was induced by pAxCAHGF plus U0126. CONCLUSIONS: Autocrine expression of HGF with signal transduction inhibitors can regulate proliferation of OC/CDE22 hepatic oval cells. In addition, the AKT pathway is important for HGF-stimulated hepatic oval cell proliferation.