The possible role of TNF-α and IL-2 in inducing tumor-associated metabolic alterations

This study was conducted to investigate the role of tumor necrosis factor- (TNF-) and interleukin-2 (IL-2) in inducing cancer cachexia, and the results were compared with those obtained from our previous study on Fisher 344 rats with methylcholanthrene-induced sarcoma. Three groups of male Fisher 344 rats received one of the following regimens: 4×10⁴ IU of human recombinant TNF- per rat per day subcutaneously (sc) for 5 consecutive days (n=5), 3.5×10⁵ U human recombinant IL-2 per rat per day sc for 14 consecutive days (n=5), or normal saline (n=5). The activities of both phosphoenolpyruvate carboxykinase (PEPCK) and malic enzyme (ME) were increased slightly in the IL-2 group. Furthermore, LPL activity was significantly increased in the adipose tissue of the TNF group and in the cardiac muscle of the IL-2 group, but not in that of the TNF group. These results show that there is a significant difference between the metabolic alterations seen in the tumor-bearing state and those induced by either TNF- or IL-2 alone. Thus, it is unlikely that IL-2 or TNF- is the sole mediator of cancer cachexia in this tumor and rat model.     

Video-assisted Thoracoscopic Lobectomy Achieves a Satisfactory Long-term Prognosis in Patients with Clinical Stage IA Lung Cancer

We designed a prospective trial to determine the long-term prognosis of video-assisted thoracoscopic (VATS) lobectomy versus conventional lobectomy for patients with clinical stage IA (T1N0M0) lung cancer. Between January 1993 and June 1994, 100 consecutive patients with clinical stage IA non-small cell lung carcinoma underwent either conventional lobectomy through an open thoracotomy (open group; n= 52) or VATS lobectomy (VATS group; n= 48). Lymph node dissections were performed in a similar manner in both groups. No significant differences were observed in the number of dissected lymph nodes between the 2 groups. Pathologic N1 and N2 disease was found in 3 and 1 patients, respectively, from the open group, and in 2 and 1 patients, respectively, from the VATS group. During the follow-up period, distant metastases and local or regional recurrences developed in 7 and 3 of the open group patients, respectively, and in 2 and 3 of the VATS group patients, respectively. Two and one of the open and VATS group patients developed second primary cancers, respectively. The overall survival rates 5 years after surgery were 85% and 90% in the open and VATS groups, respectively (log-rank test, p= 0.74; generalized Wilcoxon test, p= 0.91). VATS lobectomy with lymph node dissection achieved an excellent 5-year survival, similar to that achieved by the conventional approach.     

No Improvement of Adult Height in Non-growth Hormone (GH) Deficient Short Children with GH Treatment

It is still in doubt whether the standard-dose growth hormone (GH) used in Japan (0.5 IU/kg/week, 0.167 mg/kg/week) for growth hormone deficiency is effective for achieving significant adult height improvement in non-growth hormone deficient (non-GHD) short children. We compared the growth of GH-treated non-GHD short children with that of untreated short children to examine the effect of standard-dose GH treatment on non-GHD short children. GH treatment with recombinant human growth hormone (rhGH) was started before the age of 11 yr in 64 boys and 76 girls with non-GHD short stature registered at the Foundation for Growth Science who have now reached their adult height. In 119 untreated boys and 127 untreated girls whose height standard deviation score (SDS) was below –2 SD at the age of 6 yr, height growth was followed until 17 yr. Height SDS was significantly lower before GH treatment in the GH-treated group than at the age of 6 yr in the untreated group, in both sexes. Adult height and adult height SDS were significantly greater in the untreated group than in the GH-treated group, in both sexes, although the change in height SDS did not differ significantly. Height SDS was significantly lower before GH treatment in the GH-treated group than at the age of 6 yr in the untreated group, so 57 boys and 57 girls whose height SDS at the age of 6 yr in the untreated group closely matched the height SDS before GH treatment in the GH-treated group were chosen for comparison. Height SDS did not differ significantly between the GH-treated group before GH treatment and the untreated group at the age of 6 yr, nor were there differences between these subgroups in adult height, adult height SDS, or height SDS change, in either sex. The effect of GH treatment is reported to be dose-dependent and doses over 0.23 mg/kg/week are reported to be necessary to improve adult height in non-GHD short children. Currently, the GH dose is fixed at 0.175 mg/kg/week in Japan, and we expected to find, and indeed concluded, that ordinary GH treatment in Japanese, non-GHD short children does not improve adult height.     

Effect of promoter methylation of the p16 gene on phosphorylation of retinoblastoma gene product and growth of hepatocellular carcinoma cells.

The biological significance of hypermethylation of p16 gene promoter in human hepatocellular carcinoma (HCC) cells remains to resolved. In order to clarify the significance of methylation of p16 gene promoter, we examined the methylation status of p16 gene in association with phosphorylation of retinoblastoma gene product (pRb) and cell growth in human HCC cell lines. The presence of methylation was examined by methylation-specific PCR. Expression and phosphorylation of p16 and pRb were examined by Western blot analysis. Genetic changes were analyzed by multiplex PCR and DNA sequencing. The effect of demethylation of p16 was assessed by cell growth. p16 gene promoter was methylated in HuH7 and HLF cells. The demethylating agent, 5-aza-2-deoxycytidine (5-Aza-CdR), upregulated p16 mRNA in HuH6 and HuH7 cells. 5-Aza-CdR increased p16 protein expression in HuH6, HuH7, and HLF cells, and it clearly decreased the phosphorylation level of pRb in HuH6, HuH7 and PLC/PRF/5 cells. Treatment with 5-Aza-CdR inhibited the growth of HuH7 cells. Homozygous deletion and significant mutations were absent. Methylation in the p16 promoter region is biologically significant, being associated with phosphorylation of pRb and cell growth in human HCC cells.     

Identification of transforming activity of free fatty acid receptor 2 by retroviral expression screening

Gallbladder cancer (GBC) is a highly fatal malignancy in humans. Genetic alterations in KRAS or TP53 as well as overexpression of ERBB2 have been shown to contribute to the development of certain types of GBC. However, many cases of GBC do not harbor such genetic changes, with other transforming events awaiting discovery. We here tried to identify novel cancer-promoting genes in GBC, with the use of a retroviral cDNA expression library. A retroviral cDNA expression library was constructed from a surgically resected clinical specimen of GBC, and was used to infect 3T3 fibroblasts in a focus formation assay. cDNA incorporated into the transformed foci was rescued by PCR. One such cDNA was found to encode free fatty acid receptor 2 (FFAR2), a G protein-coupled receptor for short-chain fatty acids. The oncogenic potential of FFAR2 was confirmed both in vitro with the focus formation assay and by evaluation of cell growth in soft agar as well as in vivo with a tumorigenicity assay in nude mice. The isolated FFAR2 cDNA had no sequence alterations, suggesting that upregulation of FFAR2 expression may contribute to malignant transformation. Indeed, all of quantitative RT-PCR, in situ hybridization, and immunohistochemical analyses showed that the amount of FFAR2 mRNA and its protein product was increased in digestive tract cancer specimens. Furthermore, short-chain fatty acids potentiated the mitogenic action of FFAR2 in 3T3 cells. Our data thus, for the first time, implicate FFAR2 in carcinogenesis of the digestive tract. ( Cancer Sci 2009)     

Simple and rapid analysis of aristolochic acid contained in crude drugs and Kampo formulations with solid-phase extraction and HPLC photodiode-array detection

Simple and rapid analysis of aristolochic acid (AA) in crude drugs and Kampo extracts using a solid-phase extraction method and HPLC-PDA analysis was investigated. Extraction of AA from samples was accomplished by adding methanol containing 1% ammonia. The addition of ammonia ionized the AA of acidic substances so that they adhered to an acrylamide copolymer of a strong anion exchange resin (Sep-Pak QMA) coupled to diol silica easily. Furthermore, a mixture of acetonitrile–water–phosphoric acid (75:25:2, v/v) was effective in isolating AA from its carrier. Since almost all interfering peaks originating from contaminants in crude drugs and Kampo extract formulations could be removed, a satisfactory HPLC chromatogram of AA was obtained. A good result was also obtained when Aristolochiaceae and crude drugs containing AA were tested. Particularly in the case of the medicinal parts of Asarum, several interfering peaks and a ghost peak detected near the AA peak were eliminated. The AA contents of two Kampo extract formulations, tokishigyakukagoshuyushokyoto and ryutanshakanto, were calculated by HPLC analysis. The AA content (the sum of AA-I and AA-II) was 1.25–6.13 mg per daily dose. From an additional recovery experiment for Kampo formulations, high recovery rates of AA were obtained. Neither LC/MS nor special instrumentation was necessary. Our results suggest that this simple, quick, and sensitive analytical method to detect AA in crude drugs and Kampo extract formulations would be valuable in safety inspections of AA in crude drugs and their products.