Biomedical subjects

K Nishiguchi

Publications and source records attributed to K Nishiguchi.

At least 19 recordsLinked to original sources

PubMed2004

Laparoscopic approach for early gastric cancer within a paraesophageal hiatal hernia.

We present a case of early gastric cancer located in gastric volvulus associated with paraesophageal hiatal hernia. Two lesions of EGC were diagnosed in the distal third of the stomach, most of which had herniated into the left chest through a large hiatal defect in an organoaxial fashion. Routinely, laparoscopic-assisted distal gastrectomy (LADG) is our preferred approach for EGC, and the presence of hiatal hernia in this case did not alter our approach. Laparoscopic repair of hiatal hernia was performed successfully followed by LADG. A review of the literature supports a minimally invasive approach for both procedures and shows it to be safe, effective, and technically feasible. Further, LADG is shown to be oncologically adequate in terms of tumor margins and lymph node dissection, but its relevance to long-term disease-free survival still needs to be studied in well-designed prospective trials.

Gastrectomy↗

PubMed2001

Comparative evaluation of surgical stress of laparoscopic and open surgeries for colorectal carcinoma.

PURPOSE: To objectively evaluate the benefits of laparoscopic procedures for colorectal carcinoma, a prospective study to measure the stress response to laparoscopic surgery (n = 15) compared with open surgery (n = 12) was undertaken. In addition, to compare the various parameters relevant to surgical stress, the major surgery group (transthoracic esophagectomy for esophageal carcinoma, n = 4; and left upper abdominal evisceration for gastric carcinoma, n = 3) was assigned. METHODS: Peripheral blood samples were obtained to measure serum interleukin-6, C-reactive protein, peripheral leukocytes, and lymphocyte counts. Additionally, the level of lymphocyte apoptosis was quantified using flow cytometry. RESULTS: The interleukin-6 and C-reactive protein levels were significantly greater in the open group than in the laparoscopy group one day (P < 0.05) and two days (P < 0.05) after surgery, respectively. In the laparoscopy group, lymphocyte counts were significantly higher than in the open group two days after surgery. The laparoscopy and open groups did not differ significantly in their lymphocyte apoptotic index. In the major surgery group, the apoptotic index was significantly higher than in either the laparoscopy group or the open group in the early postoperative period. CONCLUSION: Changes in the various parameters pertinent to surgical stress evaluated in this study suggest that laparoscopic surgery for colorectal carcinoma leads to less postoperative stress than conventional open surgery.

Aged↗

PubMed2001

Efficacy of intraperitoneal and intravenous chemotherapy and left upper abdominal evisceration for advanced gastric cancer.

BACKGROUND: The study was carried out to evaluate the efficacy of intraperitoneal (i.p.) and intravenous (i.v.) chemotherapy, as well as left upper abdominal evisceration (LUAE), for patients with advanced gastric cancer. METHODS: We carried out a retrospective study of 348 patients who underwent gastrectomy for advanced gastric carcinoma between 1978 and 1998 at our institution and who had macroscopic type 3 or 4 cancer (Japanese classification) with depth of invasion to the serosal surface, but no liver metastasis or lymph node metastasis around the abdominal aorta. Cumulative survival rates were compared in patients who underwent gastrectomy together with: (1) intraoperative i.p. chemotherapy alone, (2) postoperative i.v. chemotherapy alone, (3) both i.p. and i.v., or (4) no chemotherapy. Then patients were stratified according to the presence of peritoneal dissemination (P+) and its absence (P-). In P+ patients, survival was compared between those who received i.v. chemotherapy and those who did not, and between those who received i.p. chemotherapy and those who did not. Then, survival was compared between patients with high and low immunosuppressive acidic protein (IAP) levels. Finally, we compared cumulative survival rates in patients (stratified as P+ and P-) who underwent LUAE with cumulative survival rates in those who underwent total gastrectomy combined with resection of the pancreatic body, tail, and spleen (PS). RESULTS: For P- patients, there was no survival advantage with adjuvant i.p. or i.v. therapy when compared with surgery alone. For P+ patients, however, there was an improvement in survival when patients received both i.p. and i.v., compared with survival with surgery alone (P < 0.05). In P+ patients aged less than 60 years, there was improvement in survival for those who underwent i.p. therapy together with surgery (P < 0.05), but not for those who had i.v. chemotherapy after surgery. When LUAE was examined, there was a survival advantage for this procedure when there was no peritoneal dissemination. Four long-term survivors (surviving for more than 5 years) were identified in our study. Three of the 4 patients were aged less than 60 years, and all 4 had macroscopic type 4 gastric cancers. CONCLUSION: Although the prognosis for patients with invasive type gastric cancer remains poor, there have been a few long-term survivors, in whom this survival was associated with aggressive combination therapy, including surgery, i.p., and i.v. therapy. P+ patients aged less than 60 years and patients with type 4 gastric cancer may stand to benefit most from such therapy. For P- patients, the role of adjuvant i.p. or i.v. therapy continues to be ambiguous, although LUAE in this population may be superior to PS.

Adult↗

PubMed2001

CYP2C19 genotype and pharmacokinetics of three proton pump inhibitors in healthy subjects.

PURPOSE: To predict the CYP2C19 genotype-dependence in anti-Helicobacter pylori (H. pylori) therapy when lansoprazole or rabeprazole was used instead of omeprazole as a proton pump inhibitor (PPI). METHODS: A comparative pharmacokinetic study with each PPI was designed as an open, randomized, and crossover study of 18 Japanese healthy volunteers who were classified into the homozygous, heterozygous extensive metabolizer and the poor metabolizer based on the CYP2C19 genotype determined by PCR-RFLP method. Each subject received a single oral dose of 20 mg omeprazole, 30 mg lansoprazole, or 20 mg sodium rabeprazole, with at least 1 week washout period between treatments. Plasma concentrations of PPIs and their metabolites were monitored until 12 h after medication. RESULTS: Pharmacokinetic profiles of omeprazole and lansoprazole were well correlated with the CYP2C19 genotype. The heterozygous extensive metabolizer was slightly different from the homozygote, but there was no statistically significant difference. The CYP2C19 genotype dependence found for lansoprazole was not obvious compared with omeprazole. As for rabeprazole, the pharmacokinetic profile was independent of the CYP2C19 genotype. CONCLUSIONS: CYP2C19 genotype dependence will be found in the anti-H. pylori therapy even when lansoprazole is used as the PPI.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed2001

Beta2-adrenergic receptor genotype-related changes in cAMP levels in peripheral blood mononuclear cells after multiple-dose oral procaterol.

PURPOSE: To evaluate the beta2-adrenergic receptor (beta2AR) genotype frequency in the Japanese population and the relationship between beta2AR genotype at amino acid position 16 (beta2AR-16) and desensitization to beta2-agonist ex vivo. METHODS: The beta2AR genotypes at amino acid positions 16, 27, and 164 of 92 healthy Japanese subjects were determined by polymerase chain reaction-restriction fragment-length polymorphism. The relationship between the beta2AR-16 genotype and the desensitization to beta2-agonist was examined in 10 male subjects ex vivo. Procaterol tablet (HCl salt, 50 microg, Meptin) was given orally for 5 days, and peripheral blood was obtained before and after 5 days of consecutive medications followed by the assessment of the intracellular cAMP levels in peripheral blood mononuclear cells after incubation with or without procaterol hydrochloride (0-1000 ng/mL). RESULTS: Allele frequency was Arg16:Gly16 = 46%:54%, Gln27: Glu27 = 92%:8%, and Thr164:Ile164 = 100%:0%, respectively. The cAMP levels were increased by incubation with procaterol hydrochloride, and the increase was suppressed after 5 days of consecutive medications. The suppression was more significant in the homozygote for Gly16 than the homozygote for Arg16. CONCLUSIONS: The desensitization to beta2-agonist was associated more frequently with the mutation at beta2AR-16 (Gly16).

Administration, Oral↗

PubMed2001

Effect of serum triglyceride concentration on the fluctuation of whole blood concentration of cyclosporin A in patients.

The methodology to distinguish the patients showing considerable fluctuation of the whole blood concentration of cyclosporin A (CYA) was investigated from a viewpoint of laboratory test values. First, we retrospectively examined the CYA trough blood concentrations monitored continuously. The patients were classified into three groups by the fluctuation of CYA trough blood concentrations during the examination period (Cmax/Cmin): Group 1 (Cmax/Cmin=100-200%; n=21), Group 2 (Cmax/Cmin=200-300%; n=25), and Group 3 (Cmax/Cmin=more than 300%; n=32). In the laboratory tests examined, the serum triglyceride concentrations were considerably different among the groups, and it was the highest in Group 3. Next, to elucidate the effect of serum triglyceride concentration on the CYA blood concentration, in vivo pharmacokinetic studies after single intravenous or repetitive oral administration of CYA were conducted in the model rats with pseudo-hypertriglyceridemia, hypocythemia, and acute renal failure. Only in pseudo-hypertriglyceridemia rats, the CYA blood concentration after a single intravenous injection was significantly higher than that in normal rats because of the restriction of CYA distribution to the extravascular tissues. On the other hand, the increase in the serum triglyceride concentration did not affect the fluctuation of CYA trough blood concentration after repetitive oral administration. Taken together, the fluctuation of CYA trough blood concentrations observed in the clinical situation could be due to the fluctuation of serum triglyceride concentration, and the patients with such fluctuation of serum triglyceride concentrations might also be distinguishable by the higher concentration of serum triglyceride in laboratory tests.

Acute Kidney Injury↗

PubMed2001

[A case of stage IV gastric cancer with multiple liver metastases and carcinomatous ascites responding to TS-1 for six months before progression].

We report the case of a 72-year-old male with Stage IV gastric cancer accompanied by multiple liver metastases and carcinomatous ascites which responded to chemotherapy using TS-1. Treatment of the patient with daily oral administration of 120 mg TS-1 for 4 weeks resulted in complete regression of the liver metastases and ascites, as well as a 35% reduction in the size of the primary lesion. After 2 cycles, the primary tumor size was reduced to 55% and serum CA19-9 and CA125 levels were decreased to the normal ranges. This regimen was effective without any adverse effects, and improved the patient's QOL, for 6 months before progression of liver lesions. The patient received chemotherapy at our outpatient clinic for 10 months after the first treatment, after which he died of peritonitis carcinomatosa. The current case suggests that TS-1 may have a potent therapeutic efficacy in advanced gastric cancer patients.

Aged↗

PubMed2000

The novel anticancer drug KRN5500 interacts with, but is hardly transported by, human P-glycoprotein.

The interaction of the novel anticancer drug KRN5500, a spicamycin derivative, with human P-glycoprotein (P-gp) was analyzed from the viewpoint of cellular pharmacokinetics, i.e. by means of [3H]azidopine photoaffinity labeling, cellular accumulation and transcellular transport experiments. In this study, P-gp-overexpressing LLC-GA5-COL150 cells, porcine kidney epithelial LLC-PK1 cells transformed with human MDR1 cDNA, were used, since this cell line constructs monolayers with tight junctions, and would provide sufficient information for analyzing the cellular pharmacokinetics. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the growth-inhibitory effect of KRN5500 in LLC-GA5-COL150 cells was comparable to that in LLC-PK1 cells (IC50 = 79.4 and 72.7 nM, respectively), but the inhibition of [3H]azidopine binding by KRN5500 was concentration-dependent in the membrane fraction of LLC-GA5-COL150 cells. The cellular accumulation of [14C]KRN5500 after its basal application in LLC-GA5-COL150 cells was slightly lower than that in LLC-PK1 cells, and was restored by the multidrug resistance (MDR) modulator SDZ PSC 833. The basal-to-apical transport of [14C]KRN5500 in LLC-GA5-COL150 cells was also slightly higher than that in LLC-PK1 cells, and was inhibited by SDZ PSC 833. However, the basal-to-apical transport of [14C]KRN5500 in LLC-GA5-COL150 cells was only a little higher than the apical-to-basal transport. Consequently, these results demonstrated that KRN5500 interacted with, but was hardly transported via, P-gp. These observations suggested that KRN5500 may be useful even for the treatment of tumors exhibiting P-gp-mediated MDR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Impact of the expression of cyclin-dependent kinase inhibitor p27Kip1 and apoptosis in tumor cells on the overall survival of patients with non-early stage gastric carcinoma.

BACKGROUND: The expression of p27Kip1 and apoptosis have been implicated in tumor aggressiveness and proved to be prognostic predictors for several human malignancies. In this study, the authors sought to investigate the expression of p27Kip1 and apoptosis and their potential significance in determining the prognosis of patients with non-early stage gastric carcinoma. METHODS: Primary gastric tumor specimens from 225 patients were investigated by immunohistochemistry with anti-p27Kip1 and anti-Ki-67 antibodies, and their apoptotic indices were determined with the use of an Apop-Tag in situ detection kit. RESULTS: The median p27Kip1 labeling index (LI) was 48.4%. There was a significant association between the p27 LIs and the apoptotic indices (Als). However, there was no association between the p27 LIs and the Ki-67 LIs. p27 LI was demonstrated to be one of the most significant and independent prognostic factors in multivariate analysis. Although AI was found to be prognostically significant in univariate analysis, it failed to retain an independent and significant value regarding overall survival in multivariate analysis. CONCLUSIONS: Decreased expression of p27Kip1 and reduction of apoptotic potential were two of the most important factors in predicting a poor prognosis for patients with non-early stage gastric carcinoma. These findings support the hypothesis that decreased p27Kip1 expression, which may reflect a decreased rate of apoptosis, is closely related to the aggressiveness of gastric carcinoma. Therefore, the assessment of p27Kip1 expression and apoptotic potential may prove valuable in identifying patients with gastric carcinoma who are at high risk for recurrence and would benefit from adjuvant therapy.

Adenocarcinoma↗

PubMed1999

Clinical evaluation of lymph node metastasis in gastric cancer defined by the fifth edition of the TNM classification in comparison with the Japanese system.

BACKGROUND: This study compared the classification of lymph node metastasis according to the number of involved nodes based on the new tumour node metastasis (TNM) system (fifth edition) with the classification by the Japanese Research Society for Gastric Cancer from an anatomical perspective. METHODS: The two classifications were related to long-term results in 1489 patients with gastric cancer who underwent gastrectomy with systematic extended lymphadenectomy. RESULTS: Both classifications performed well as prognostic indicators (5-year survival rates: pathological (p) N0, 89 per cent; pN1, 66 per cent; pN2, 34 per cent; pN3, nil; and M1, 10 per cent by the TNM classification; n0, 89 per cent; n1, 63 per cent; n2, 46 per cent; n3, 20 per cent; and n4, 8 per cent by the Japanese classification). For regional lymph nodes, the TNM classification was a better index of the prognosis. Significant survival differences were observed among patients with M1 disease according to the number of involved lymph nodes (between one and six nodes, 48 per cent; seven to 15 nodes, 12 per cent; more than 15 nodes, 2 per cent), indicating that patients with distant metastatic lymph nodes (M1) should also be classified by the number of involved nodes. On the other hand, the Japanese classification has the added benefit of being a good indicator of the anatomical extent of lymphadenectomy. CONCLUSION: The new TNM classification provided a better index of the prognosis of patients who underwent systematic lymph node dissection. However, both classifications have specific benefits in the surgical treatment of gastric cancer.

Female↗

PubMed1999

Cellular pharmacokinetic aspects of reversal effect of itraconazole on P-glycoprotein-mediated resistance of anticancer drugs.

The reversal effect of itraconazole on P-glycoprotein (P-gp)-mediated resistance of vinblastine, daunorubicin and doxorubicin was analyzed from a cellular pharmacokinetic point of view, namely by [3H]azidopine photoaffinity labeling, intracellular accumulation and transcellular transport experiments. The LLC-GA5-COL150 cells, which expressed human P-gp selectively on the apical membrane due to transfection of MDR1 cDNA into the porcine kidney epithelial cells (LLC-PK1 cells), was used here, since this cell line constructs the monolayer with tight junction, being able to characterize the cellular pharmacokinetics. In LLC-GA5-COL150 cells, itraconazole caused a reversal from resistance as shown by a growth inhibition assay. [3H]Azidopine photoaffinity labeling demonstrated that itraconazole, vinblastine, daunorubicin and doxorubicin showed higher binding ability for P-gp compared with digoxin, suggesting the following results were via P-gp. The intracellular accumulation of [3H]vinblastine, [3H]daunorubicin and [14C]doxorubicin after their application on the basal and apical sides was increased by itraconazole. These changes were similar to the dose modifying factors determined by the growth inhibition assay. However, their basal-to-apical transport was hardly affected by itraconazole, and this was explained by the fact that itraconazole inhibited P-gp, and subsequently increased their intracellular concentration and then the non-P-gp mediated transport from the intracellular space to apical side. The apical-to-basal transport of [3H]vinblastine, [3H]daunorubicin and [14C]doxorubicin was increased by itraconazole, and this was reasonably explained by the inhibition of P-gp, and partly also by the increase of their intracellular concentration via the inhibition of P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Effects of secretable SOD delivered by genetically modified cells on xanthine/xanthine oxidase and paraquat-induced cytotoxicity in vitro.

We designed a new eukaryotic expression vector for secretable superoxide dismutase (SOD), which expresses human SOD cDNA by fusing it to 1 connecting amino acid and the signal peptide DNA sequence of the human interleukin-2 (IL-2) gene (IL-SOD(2) cDNA). The ILSOD(2) cDNA constructed by PCR-based gene expression was ligated into the multicloning site of the pRc/CMV plasmid (pRc/CMV-ILSOD(2)). Rat lung epithelial-like cells (L2 cells) and rat skin fibroblasts (FR cells) were transfected with pRc/CMV-ILSOD(2) by lipofection. The extracellular SOD activities of IS(2)-L2 cells (L2 cells transfected with pRc/CMV-ILSOD(2)) and IS(2)-FR cells (FR cells transfected with pRc/CMV-ILSOD(2)) were 2-3 times higher than those of host cells. Initially, we investigated the protective effect of extracellular SOD secreted from these transformed cells (IS(2)-L2 and IS(2)-FR cells) on extracellular superoxide anion (xanthine/xanthine oxidase; X/XO treatment)-induced cytotoxicity in normal cells. The sensitivities of these transformed cells to X/XO-induced cytotoxicity was decreased significantly as compared with that of host cells. Although, the conditioned medium from IS(2)-L2 and IS(2)-FR cells protected against X/XO-induced cytotoxicity, the conditioned medium from host cells (L2 and FR cells) showed no significant effects on X/XO-induced cytotoxicity. Furthermore, the conditioned medium from transformed cells was more effective than that of host cells against lipid peroxidation by normal cells under conditions of oxidative stress. Second, we generated superoxide anions in the intracellular space by paraquat treatment. The transformed cells were more sensitive to paraquat-induced cytotoxicity than host cells. Following addition of catalase, the sensitivity of these genetically modified cells to paraquat became equivalent to that of host cells. These results indicated a protective effect of transfection with secretable SOD genes against extracellular superoxide anion-induced cytotoxicity although no such protective effect was observed against the intracellular cytotoxicity generated by paraquat treatment.

Animals↗

PubMed1998

Effects of Cu, Zn-superoxide dismutase delivered by genetically modified skin fibroblasts on cold-induced skin edema in rats.

The effects of Cu, Zn-superoxide disumutase (SOD) delivered by genetically modified skin fibroblasts on cold-induced skin edema were studied in rats. Cold-induced skin edema was induced on the dorsal skin following transplantation of ILSOD cells, genetically modified skin fibroblasts which release secretable SOD protein into the extracellular space. The degree of skin edema induced by cold injury was estimated by measuring the amounts of Evans' blue (EB) leaking into the injured skin following intravenously administration. The amounts of EB leakage were significantly reduced by transplantation of ILSOD cells relative to that observed following transplantation of host cells as a control. The degrees and durability of these effects of ILSOD cells were dependent on the number of cells transplanted. Also, the increases of lipid peroxidation following cold injury were significantly reduced by transplantation of ILSOD cells but not of host cells. These findings suggested that transplantation of ILSOD cells was a suitable delivery system for obtaining efficient and continuous effects of SOD. This strategy using genetically modified skin fibroblasts may also be useful as a drug delivery system for other therapeutic proteins.

Animals↗

PubMed1998

Prognostic value of tumor cell proliferation and intratumor microvessel density in advanced gastric cancer treated with curative surgery.

To clarify the prognostic value of tumor cell proliferation [proliferating cell nuclear antigen (PCNA)-positive rate assessed by anti-PCNA immunostaining] and intratumor microvessel density (/0.74 mm2, assessed by anti-CD34 immunostaining) in gastric cancer, 192 advanced gastric cancer patients who underwent curative surgery were investigated. Multivariate analysis showed that the following variables were significantly related to prognosis: age (P=0.029), depth of invasion (P=0.005), lymph node metastasis (P=0.006), lymphatic invasion (P=0.038), venous invasion (P=0.0005), PCNA-positive rate (P=0.049) and intratumor microvessel density (P=0. 011). In conclusion, tumor cell proliferation and intratumor microvessel density were independent prognostic factors in patients with advanced gastric cancer undergoing curative surgery.

Adult↗

PubMed1997

Enhanced anti-inflammatory effects of Cu, Zn-superoxide dismutase delivered by genetically modified skin fibroblasts in vitro and in vivo.

PURPOSE: The purpose of this work was to evaluate the anti-inflammatory effects of secretable human Cu, Zn-superoxide dismutase (hSOD) delivered by genetically modified skin fibroblasts in vitro and in vivo. METHODS: Rat skin fibroblasts were transfected with pRc/CMV-ILSOD including secretable SOD-coding cDNA. The effects of host and transformants on oxidative stress in vitro models using the xanthine/xanthine oxidase (X/XO) system were examined to study the paracrine SOD action. The anti-inflammatory effects by transplantation of host and transformants were evaluated in an acute inflammation model, carrageenin-induced paw edema, in rats. RESULTS: The transformants (ILSOD cells) secreted SOD protein into the extracellular space, and the extracellular SOD activity in ILSOD cells cultures was significantly increased compared with that in host cell cultures. ILSOD cells diminished the cytotoxic activity by X/XO in a paracrine fashion. These protective effects of ILSOD cells against X/XO-induced cytotoxicity correlated well with the decrease in lipid peroxidation in the damaged cells. The in vivo study showed that transplantation of ILSOD cell suspensions into the hind paw in rats inhibited carrageenin-induced paw edema for at least 7 days, and the degree and the durability of these inhibitory effects were dependent on the number of ILSOD cells transplanted. These inhibitory effects of ILSOD cell suspensions were reduced by co-administration of antiserum for hSOD. Furthermore, the healing of paw edema caused by carrageenin was markedly enhanced by transplantation of ILSOD cells into the edemics hind paw. CONCLUSIONS: The findings suggested that genetically modified skin fibroblasts are a suitable delivery system for obtaining an efficient and continuous supply of SOD to the target site, and this strategy may be a useful drug delivery system for therapeutic proteins.

Animals↗

PubMed1997

Protective effect of transfection with secretable superoxide dismutase (SOD) (a signal sequence-SOD fusion protein coding cDNA) expression vector on superoxide anion-induced cytotoxicity in vitro.

For ex vivo gene therapy, superoxide dismutase (SOD) must be secreted into the extracellular space and delivered to damaged cells. Recombinant DNA technique can be used to produce a secretory protein that is fused to a non-secretory protein and a signal peptide of another secretory protein gene. We constructed a secretable SOD eukaryotic expression vector which expresses human SOD cDNA by fusing it to the signal peptide DNA sequence of the human interleukin-2 (IL-2) gene. The ILSOD cDNA constructed by PCR-based gene expression was ligated into the multicloning site of the pRc/CMV plasmid (pRc/CMV-ILSOD). Rat lung epithelial like cells (L2 cells) were transfected with pRc/CMV-ILSOD by lipofection. The extracellular SOD activity of ILSOD-L2 cells (transfected cells with pRc/CMV-ILSOD) was 3 times as high as that of host cells. We used the xanthin (X)/xanthin oxidase (XO) system to produce superoxide anions at the extracellular space. We initially investigated the direct cytotoxicity of superoxide anions upon cells. Host and ILSOD-L2 cells were killed by using X/XO, although the sensitivity of the ILSOD-L2 cells to X/XO induced cytotoxicity was significantly decreased compared with that of host cells. The production of lipid peroxidated substances in the host in the presence of X/XO increased to about twice the control (absence of X/XO) level. However, that of ILSOD-L2 cells did not change in the presence of X/XO. Therefore, ILSOD-L2 cells were resistant to X/XO induced lipid peroxidation. These findings indicated that ILSOD gene transfection protected against direct oxidant stress by X/XO. We then investigated the effect of extracellular SOD secreted from ILSOD-L2 cells on extracellular superoxide anion induced cytotoxicity in normal cells. The conditioned media of host cells had no significant effect upon X/XO induced cytotoxicity. However, the conditioned media of ILSOD-L2 cells protected against X/XO induced cytotoxicity. Furthermore, the conditioned medium of ILSOD-L2 cells was more effective than that of host cells against the production of lipid peroxidated substances by normal cells under conditions of oxidative stress. These results indicated that non-secretable protein could be delivered to target cells by means of DNA engineering. This strategy could thus provide an ex vivo means of applying gene therapy using non-secretable proteins.

Animals↗

PubMed1996

Effects of transfection with the Cu, Zn-superoxide dismutase gene on xanthine/xanthine oxidase-induced cytotoxicity in fibroblasts from rat skin.

PURPOSE: The effects of transfection with the human Cu, Zn-superoxide dismutase (hSOD)4 gene on active oxygen-induced cytotoxicity in rat skin fibroblasts (FR) were studied for the purpose of developing the novel delivery system of hSOD using hSOD gene. METHODS: An expression plasmid for hSOD, pRc/RSV-SOD, was constructed and used to transfect FR cells. Xanthine (X)/xanthine oxidase (XO) system were used to generate active oxygen species. The effects of transfection with the hSOD gene on active oxygen-induced cytotoxicity were assessed by comparing the number of surviving cells and the level of lipid peroxidation in host and transformants after exposure to X/XO system. RESULTS: The cellular SOD activity in RSV-SOD cells transfected with pRc/RSV-SOD was significantly increased in comparison with host or RSV cells transfected with the pRc/RSV plasmid containing no hSOD gene as a control. Furthermore, Western blot analysis using an anti-hSOD antibody indicated the production of hSOD in RSV-SOD cells. On the other hand, although the numbers of surviving cells in both host and RSV-SOD cultures after exposure to X/XO system decreased in a time-dependent manner, the decrease in number of surviving RSV-SOD cells was less than that in host cells. In the presence of catalase, the decreases in number of surviving cells in both host and RSV-SOD cultures after exposure to the X/XO system were also less than those in the absence of catalase. However, the decreases in cell survival in RSV-SOD cultures were significantly less than those in host cells in the presence of catalase. Furthermore, the levels of lipid peroxidation in RSV-SOD cells exposed to the X/XO system in the presence or absence of catalase were lower than those in host cells. These results indicated that the increase in cellular SOD activity by transfection with the hSOD gene protects cells from oxidative stress. CONCLUSIONS: Human SOD gene therapy may be useful for treatment of diseases in which oxidative tissue damage is produced.

Analysis of Variance↗

PubMed1996

Pharmaceutical studies for gene therapy: expression of human Cu, Zn-superoxide dismutase gene transfected by lipofection in rat skin fibroblasts.

To evaluate whether lipofection using Lipofectin is suitable for delivering foreign genes into skin fibroblasts as target cells, we performed experiments using human superoxide dismutase (hSOD) and neomycin-resistance (Neo) genes as models in rat skin fibroblasts (FR and primary cells) in vitro. The amounts of DNA used in the lipofection procedure significantly affected the transfection efficiencies, and the optimal amounts were determined for all cells used. However, the efficiencies in rat skin fibroblasts were about 20-fold higher than that in rat lung epithelial-like cells (L2 cells). The differences in plasmid vectors (pRc/RSV-SOD and pRc/CMV-SOD) hardly affected the transfection efficiencies. The amounts of Lipofectin significantly affected the transfection efficiencies, and the optimal amounts were determined for both types of skin fibroblasts. However, cytotoxic effects in both skin fibroblasts were observed with high doses of Lipofectin. On the other hand, with optimal amounts of DNA and Lipofectin, the reporter gene (NeoT) introduced into cells was mainly integrated into the host cell chromosome. Western blot analysis showed the continuous expression of hSOD protein for at least 45 d in skin fibroblasts transfected with the expression plasmid for hSOD by Lipofectin under the optimal conditions, and the cellular SOD activity fluctuated in parallel with the expression of hSOD protein. Differences in the type of cells also affected the expression of hSOD. These results indicate that it is necessary to set up optimal conditions for transfection using Lipofectin for each cell type, and that transfection with Lipofectin under optimal conditions may be an efficient method for introduction of foreign genes into skin fibroblasts for use as a clinical delivery system of therapeutic protein.

Animals↗