PHARMACOKINETIC STUDY OF INTRAPERITONEALLY ADMINISTERED ETOPOSIDE AGAINST PERITONITIS CARCINOMATOSA

Etoposide is becoming important in primary and salvage therapy for ovarian cancer. In the present study, we administered etoposide (100-300 mg/subject) intraperitoneally to six patients suffering from cancerous peritonitis, particularly that resulting from ovarian cancer, to investigate the bioavailability and pharmacokinetics of this drug. The peak etoposide level in the ascites was 80 μg/ml. Twelve hours after intrapertoneal administration (i. p.), more than 10 μg/ml of etoposide was still found in ascites. The serum level after administration of 100 mg i. p. reached approximately 4 μg/ml within 30 minutes, and 1 μg/ml of etoposide was still found in serum after 24 hours. The etoposide levels in ascites and serum after 300 mg i. p. were higher than those after 100 mg i. p. When the peritoneum was intact, the area under the curve (AUC) of etoposide in ascites was low (164 μg・h/ml), and the peritoneal clearance (Clp) was high. In contrast, in advanced cancerous peritonitis, the AUC in ascites was high (500 μg・h./ml) and the Clp was low. The AUC of etoposide in the ascites of patients with cancerous peritonitis was more than five-fold greater than that of patients with an intact peritoneum, while MRT (mean residence time) was 15-fold, and VRT (variance of residence time) was 300-fold greater. The AUC ratio in intact peritoneum was 4.1, and that in cancerous peritonitis ranged from 17.8 to 27.1. AUC, MRT and VRT of etoposide transported into the blood were slightly higher in advanced cases than in those with intact peritoneum. These findings indicate that intraperitoneal etoposide han not only a direct anticancer effect in the abdominal cavity but also shows effects via the vascular system of the tumor

IN VITRO CYTOTOXICITY OF CPT-11 (SN-38) ALONE AND IN COMBINATION WITH CISPLATIN ON OVARIAN CANCER CELLS

We investigated the in vitro cytotoxic effects of CPT-11, a new derivative of camptothecin, and its metabolizable form in vivo, SN-38, on cisplatin-resistant (SHIN-3) and -sensitive (MN-1) ovarian cancer cell lines using the MTT assay. We also attempted to identify the optimal schedule of administration for cisplatin combined with low-dose continuous exposure to CPT-11 (3 μg/ml) or SN-38 (5 ng/ml). With either CPT-11 or SN-38 alone, a marked schedule-dependent inhibition of growth was obtained with exposure for over 40 hours to concentrations of those agents applicable to clinical use (CPT-11 : <7.58 μg/ml, SN-38 : <72 ng/ml), even in a cisplatin-resistant cell line. Increasing the assay AUC of these agents only by dose escalation did not enhance cytotoxity with both MN-1 (CPT-11 : 3-50 μg/ml, SN-38 : 2.16-72 ng/ml) and SHIN-3 (CPT-11 : 5-50 μg/ml, SN-38 : 21.6-720 ng/ml) cell lines up to 72 hours of exposure. Pretreatment of SHIN-3 cells with either CPT-11 (3 μg/ml) or SN-38 (5 ng/ml) for 48 hours before the administration of cisplatin (4-20 μg/ml) appeared to produce an inhibition which exceeded that produced by either a longer (96 hours) or shorter (0 hour) pretreatment with them. Flow cytometric analysis showed that treatment with CPT-11 and SN-38 in SHIN-3 cell line was associated with a peak in G₂/M phase at 24 hours and an increase in the G₀/G₁ phase fraction for up to 96 hours. It is thus suggested that the continuous administration of a low dose of CPT-11 or of SN-38 may be useful clinically. The in vitro cytotoxicity of cisplatin could be increased by pretreatment with either CPT-11 or SN-38

Alleviation of Brain Hypoperfusion after Preventative Treatment with Lomerizine in an Elderly Migraineur with Aura

Previous studies of brain single-photon emission tomography (SPECT) showed changes of regional cerebral blood flow (rCBF) in migraineurs during prodromes or headache attacks. Little is known about how successful medication of migraine prevention can reflect rCBF in migraineurs. We highlighted alternation of brain SPECT findings in a migraineur with aura before and after prophylactic treatment with lomerizine, a calcium channel blocker. A 70-year-old man with migraine developed visual disturbance frequently at walking exercise for the recent 3 months. After this visual attack, a mild-degree of throbbing headache occured occasionally. Brain SPECT using 99mTc-ethyl cysteinate dimer was performed at interictal time of migraine. Brain SPECT before lomerizine treatment revealed hypoperfusion in the frontal, parietal, and occipital regions. He was diagnosed with recurrence of migraine with aura (MA). Lomerizine (10 mg/day, po) was administered for 3 months. MA and visual aura without headache were dramatically improved. Migraine attacks and visual disturbance were not induced at exercise. At 3 months after lomerizine medication, brain SPECT showed remarkable increase of rCBF. These SPECT changes of our patient indicated that antimigraine mechanism of lomerizine could contribute to restoration of cerebral hypoperfusion

Experimental Construction of BMP2 and VEGF Gene Modified Tissue Engineering Bone in Vitro

The purpose of this study was to investigate the feasibility and advantages of constructing a novel tissue engineering bone, using β-tricalcium phosphate (β-TCP) and rat bone marrow mesenchymal stem cells (MSCs), modified with human bone morphogenetic protein 2 gene (hBMP2) and human vascular endothelial growth factor 165 gene (hVEGF165), through lentiviral transfection. Both genes were successfully co-expressed in the co-transfection group for up to eight weeks confirmed by enzyme-linked immunosorbent assay (ELISA). After seeding MSCs onto the scaffolds, scanning electron microscopy (SEM) observation showed that MSCs grew and proliferated well in co-transfection group at 7 and 14 days. There was no significant difference among all the groups in hoechst DNA assay for cell proliferation for 14 days after cell seeding (P > 0.05), but the highest alkaline phosphatase (ALP) activity was observed in the co-transfection group at 14 days after cell seeding (p < 0.01). These results demonstrated that it was advantageous to construct tissue engineering bone using β-TCP combined with MSCs lentivirally co-transfected with BMP2 and VEGF165, providing an innovative way for treating bone defects

Mesothelial cell differentiation into osteoblast- and adipocyte-like cells

Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm including osteoblasts and adipocytes. To examine this, a functional assay of bone formation and an adipogenic assay were performed in vitro with primary rat and human mesothelial cells maintained in osteogenic or adipogenic medium (AM) for 0–26 days. Mesothelial cells expressed increasing levels of alkaline phosphatase, an early marker of the osteoblast phenotype, and formed mineralized bone-like nodules. Mesothelial cells also accumulated lipid indicative of a mature adipocyte phenotype when cultured in AM. All cells expressed several key osteoblast and adipocyte markers, including osteoblast-specific runt-related transcription factor 2, and demonstrated changes in mRNA expression consistent with epithelial-to-mesenchymal transition. In conclusion, these studies confirm that mesothelial cells have the capacity to differentiate into osteoblast- and adipocyte-like cells, providing definitive evidence of their multipotential nature. These data strongly support mesothelial cell differentiation as the potential source of different tissue types in MM tumours and other serosal pathologies, and add support for the use of mesothelial cells in regenerative therapies

Long-term cultivation of colorectal carcinoma cells with anti-cancer drugs induces drug resistance and telomere elongation: an in vitro study

BACKGROUND: The role of telomerase activation in the expression and/or maintenance of drug resistance is not clearly understood. Therefore, we investigated the relationships, among the telomerase activity, telomere length and the expression of multidrug resistance genes in colorectal cancer cell lines cultivated with anti-cancer drugs. METHODS: LoVo and DLD-1 cells were continuously grown in the presence of both CDDP and 5-FU for up to 100 days. Cell proliferation, telomerase activity, telomere length and the expression of multidrug resistance genes were serially monitored as the PDL increased. RESULTS: The expression of multidrug resistance genes tended to increase as the PDL increased. However, an abnormal aneuploid clone was not detected as far as the cells were monitored by a DNA histogram analysis. Tumor cells showing resistance to anti-cancer drugs revealed a higher cell proliferation rate. The telomere length gradually increased with a progressive PDL. The telomerase activity reached a maximum level at 15 PDL in LoVo cells and at 27 PDL in DLD-1 cells. An increase in the mRNA expression of the telomerase components, especially in hTERT and in hTR, was observed at the same PDLs. CONCLUSIONS: These results suggest that a high telomerase activity and an elongation of telomeres both appear to help maintain and/or increase drug resistance in colorectal cancer cells. Cancer cells with long telomeres and a high proliferative activity may thus be able to better survive exposure to anti-cancer drugs. This is presumably due to an increased chromosome stability and a strong expression of both mdr-1 and MRP genes

Conjugated docosahexaenoic acid suppresses KPL-1 human breast cancer cell growth in vitro and in vivo: potential mechanisms of action

Introduction The present study was conducted to examine the effect of conjugated docosahexaenoic acid (CDHA) on cell growth, cell cycle progression, mode of cell death, and expression of cell cycle regulatory and/or apoptosis-related proteins in KPL-1 human breast cancer cell line. This effect of CDHA was compared with that of docosahexaenoic acid (DHA). Methods KPL-1 cell growth was assessed by colorimetric 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; cell cycle progression and mode of cell death were examined by flow cytometry; and levels of expression of p53, p21Cip1/Waf1, cyclin D1, Bax, and Bcl-2 proteins were examined by Western blotting analysis. In vivo tumor growth was examined by injecting KPL-1 cells subcutaneously into the area of the right thoracic mammary fat pad of female athymic mice fed a CDHA diet. Results CDHA inhibited KPL-1 cells more effectively than did DHA (50% inhibitory concentration for 72 hours: 97 μmol/l and 270 μmol/l, respectively). With both CDHA and DHA growth inhibition was due to apoptosis, as indicated by the appearance of a sub-G1 fraction. The apoptosis cascade involved downregulation of Bcl-2 protein; Bax expression was unchanged. Cell cycle progression was due to G0/G1 arrest, which involved increased expression of p53 and p21Cip1/Waf1, and decreased expression of cyclin D1. CDHA modulated cell cycle regulatory proteins and apoptosis-related proteins in a manner similar to that of parent DHA. In the athymic mouse system 1.0% dietary CDHA, but not 0.2%, significantly suppressed growth of KPL-1 tumor cells; CDHA tended to decrease regional lymph node metastasis in a dose dependent manner. Conclusion CDHA inhibited growth of KPL-1 human breast cancer cells in vitro more effectively than did DHA. The mechanisms of action involved modulation of apoptosis cascade and cell cycle progression. Dietary CDHA at 1.0% suppressed KPL-1 cell growth in the athymic mouse system.</p