- Email: [email protected]
In Vitro and Vivo Effects of Extracorporeal Shock Waves on Malignant Cells
Accepted 89
90
RNA CATABOLITES AS CANCER MARKERS IN TRANSITIONAL CELL CANCER. Nicholas A. Romas,New York,NY; Irwin Clark*, James W. MacKenzie* and Lin Win*, New Brunswick,NJ. (Presentation by Dr. Romas) The present study measured 3 urinary nu~leosides (Pseudouridine (U), 7 methylguanine (m7Gua), N2 N2 dimethylguanosine (m22G)) in 18 patients with varlous clinical stages of transitional cell carcinoma of the upper and lower urinary tract. RNA catabolites i.e. modified nucleosides and bases are significantly elevated in human cancers. Following transcription from DNA, the RNAs are modified in various ways e.g. by methylation,sulfation, etc. After the RNAs have carried out their various functions in the cell, they are degraded to nucleosides and/or bases. The modified nucleosides or bases cannot be used for nucleic acid synthesis and are degraded further or excreted in the urine. One of the authors (I.C.) developed a method using high performance liquid chromatography to measure the levels of nucleosides in small amounts of biological fluids. Sixty-three controls were studied and the mean as well as standard deviation were calculated (U=20.3+ 2.75, m7Gua 2.46 + .95,m22G 1.22+ .27). An elevation was noted if a patient's urine showed 1 or more nculeoside elevated by 2 standard deviations above the mean. Twenty-six urines on 16 patients with active disease and 2 patients with clinically inactive disease were studied. Thirteen (13/16) patients had elevations and 2 cases with inactive disease were negative.
IN VITRO AND VIVO EFFECTS OF EXTRACORPOREAL SHOCK WAVES ON MALIGNANT CELLS. Randall F. Randazzo*, Hajnalka LovrekovichtSunita Bhuta* Los Angeles, CA (Presentation by Dr. Randazzo) In the in vitro experiment the effects of extra corporeal shock waves (ESW) on a human renal cell carcinoma cell line,
RCC,
and on a normal human
embryonic kidney (NHEK) cell line were examined. Using 18 kV and 100 shock/min. the suspensions were exposed to 0, 800, 1400 or 2000 shock waves (SW). All five parameters of RCC tumor cell injury which were measured, i.e, cell viability, cell attachment, electron microscopic evidence of damage and loss of tumorigenicity in Nude mice were aug-
mented with increasing SW levels. The effect was dose related. At 2000 SW a significant decrease in RCC viability, cell growth and cell attachment was seen compared to the NHEK cells (p( 0.01) Electron microscopic evaluation of RCC cells immediately after 1400 and 2000 SW treatment showed marked cell membrane disruption with loss of cell membrane processes, loss of the internal mite chondrial structure, segmentation of the nucleus with attenuation and loss of nuclear chromatin.
NHEK cells showed much less cell membrane, nuclear and mitochondrial damage, At 120 hours after ESW there was
some improvement
in ultrastructural mor-
phology in both cell types with tile 11.)iEK cells havr1;,°\hbee\tne\i";i°/Pe\0/e0ilm"ent 5 X 10 5 FANFT induced mouse bladder tumor cells were injected into the
right hind leg of C3H/he mice. The transplanted tumor was exposed to either no SW or 1400 SW. 1400 SW alone at day 12 can significantly decrease tumor size. Combining 1400 SW with cisplatin does not enhance each other's inhibitory effect.
Doxo-
rubicin and 1400 SW have a significant synergistic tumor inhibitory effect.
91
92
A DOSE RESPONSE ANALYSIS OF PURPURIN DE~IVATIVES USED AS PHOTOSENS!TIZERS FOR THE PHOTODYNAMIC TREATMENT OF TRANSPLANTABLE FANFT INDUCED UROTHELIAL TUMORS. Steven H. Selman, M.D.,*Greta M. Garbo, Ph.D.,*Rick W. Keck, B.S.,*Martha Kreimer-Birnbaum, Ph.D. and*Alan R. Morgan, Ph.D., Toledo, Ohio (Presentation to be made by Dr. Selman) Rats engrafted with transplantable N-[4-(5-nitro2-furyl)-2-thiazolyl] formamide (FANFT) induced urothelial tumors were treated with purpurin derivatives and red light (> 590 nm, 360 joules/cm2). Treated and control tumors were harvested 12 days after treatment and dry weights compared. At doses of 5.0 ug/g and 2.5 ug/g body weight, the purpurins designated NTl and NT2, when combined with light, caused statistically significant (p < 0.02) tumor regression when compared to light shielded controls. At 1.0 ug/g body weight, NTl and light also induced significant tumor regression (p < 0.02). In previously reported work using the same tumor model, hematoporphyrin derivative (HpD), a widely used tumor photosensitizer, was ineffective in causing tumor regression in doses less than 10 ug/g body weight. Purpurins, which have strong absorption bands above 650 nm and can be synthesized with a high degree of purity, appear to have potential as new photosensit i zers for photodynami c therap_y.
IN VITRO SENSITIVITY OF THREE BLADDER CARCINOMA CELL LINES TO DIFLOUROMETHYLORNITHINE AND METHYLGLYOXAL BIS-GUANYLHYDRAZONE. MUHAMMAD A. BUT,RTJL, NRW YORK, NY (PRESENTATION TO BE MADE BY DR. BULBUL.) Depletion of cellular polyamines by inhibiting biosynthesis has been demonstrated to slow growth of implanted tumors in the experimental animal. Here we report on the effect of ~-diflouromethylornithine (DFMO) an irreversible inhibitor of ornithine decaboxylase and methylglyoxal bisguanylhydrazone (MGBG) a spermidine synthesis inhibitor on tbe in vitro growth of three human bladder tumor cell lines, 2. 5x101l cells of RT4, T24 and 253J human bladder tumor lines were plated seperately on 12 well/plates with RPMI 1640+ 10% fet.al bovine serum. Wells were divided into 4 groups with group 1 serving as control. At day one cells in group 2 were exposed to l.OmM DFMO for 24 hours., cells in group 3 were exposed to 0.04mM MGBG for one hour while cells in group 4 were exposed to DFMO and MGBG sequentially. Cellular proliferation was assessed serially every two days in all groups using (3H) thymidine incorporation assay. Simultanously the levels of cellular polyamines (putrescine, spermidine and spermine) in 4 groups were determined using High Pressure Liquid Chromatography (HPLC). Twenty four hours following the addition of the drugs there was 15%, 30% and 60% inhibition of growth in groups 2,3 and 4 respectively as compared to the control group. After day 3, growth in group 4 remained significantly ()70%) and persistently inhibited while cellular proliferation in groups 2 and 3 recovered and approached that of the control by day seven. These results were consistent for all 3 cell lines with minimal variations. Interestingly the levels of putrescine, spermidine and spermine in the surviving cells, in all groups and time intervals were similar and not significantly different.
126A
90
RNA CATABOLITES AS CANCER MARKERS IN TRANSITIONAL CELL CANCER. Nicholas A. Romas,New York,NY; Irwin Clark*, James W. MacKenzie* and Lin Win*, New Brunswick,NJ. (Presentation by Dr. Romas) The present study measured 3 urinary nu~leosides (Pseudouridine (U), 7 methylguanine (m7Gua), N2 N2 dimethylguanosine (m22G)) in 18 patients with varlous clinical stages of transitional cell carcinoma of the upper and lower urinary tract. RNA catabolites i.e. modified nucleosides and bases are significantly elevated in human cancers. Following transcription from DNA, the RNAs are modified in various ways e.g. by methylation,sulfation, etc. After the RNAs have carried out their various functions in the cell, they are degraded to nucleosides and/or bases. The modified nucleosides or bases cannot be used for nucleic acid synthesis and are degraded further or excreted in the urine. One of the authors (I.C.) developed a method using high performance liquid chromatography to measure the levels of nucleosides in small amounts of biological fluids. Sixty-three controls were studied and the mean as well as standard deviation were calculated (U=20.3+ 2.75, m7Gua 2.46 + .95,m22G 1.22+ .27). An elevation was noted if a patient's urine showed 1 or more nculeoside elevated by 2 standard deviations above the mean. Twenty-six urines on 16 patients with active disease and 2 patients with clinically inactive disease were studied. Thirteen (13/16) patients had elevations and 2 cases with inactive disease were negative.
IN VITRO AND VIVO EFFECTS OF EXTRACORPOREAL SHOCK WAVES ON MALIGNANT CELLS. Randall F. Randazzo*, Hajnalka LovrekovichtSunita Bhuta* Los Angeles, CA (Presentation by Dr. Randazzo) In the in vitro experiment the effects of extra corporeal shock waves (ESW) on a human renal cell carcinoma cell line,
RCC,
and on a normal human
embryonic kidney (NHEK) cell line were examined. Using 18 kV and 100 shock/min. the suspensions were exposed to 0, 800, 1400 or 2000 shock waves (SW). All five parameters of RCC tumor cell injury which were measured, i.e, cell viability, cell attachment, electron microscopic evidence of damage and loss of tumorigenicity in Nude mice were aug-
mented with increasing SW levels. The effect was dose related. At 2000 SW a significant decrease in RCC viability, cell growth and cell attachment was seen compared to the NHEK cells (p( 0.01) Electron microscopic evaluation of RCC cells immediately after 1400 and 2000 SW treatment showed marked cell membrane disruption with loss of cell membrane processes, loss of the internal mite chondrial structure, segmentation of the nucleus with attenuation and loss of nuclear chromatin.
NHEK cells showed much less cell membrane, nuclear and mitochondrial damage, At 120 hours after ESW there was
some improvement
in ultrastructural mor-
phology in both cell types with tile 11.)iEK cells havr1;,°\hbee\tne\i";i°/Pe\0/e0ilm"ent 5 X 10 5 FANFT induced mouse bladder tumor cells were injected into the
right hind leg of C3H/he mice. The transplanted tumor was exposed to either no SW or 1400 SW. 1400 SW alone at day 12 can significantly decrease tumor size. Combining 1400 SW with cisplatin does not enhance each other's inhibitory effect.
Doxo-
rubicin and 1400 SW have a significant synergistic tumor inhibitory effect.
91
92
A DOSE RESPONSE ANALYSIS OF PURPURIN DE~IVATIVES USED AS PHOTOSENS!TIZERS FOR THE PHOTODYNAMIC TREATMENT OF TRANSPLANTABLE FANFT INDUCED UROTHELIAL TUMORS. Steven H. Selman, M.D.,*Greta M. Garbo, Ph.D.,*Rick W. Keck, B.S.,*Martha Kreimer-Birnbaum, Ph.D. and*Alan R. Morgan, Ph.D., Toledo, Ohio (Presentation to be made by Dr. Selman) Rats engrafted with transplantable N-[4-(5-nitro2-furyl)-2-thiazolyl] formamide (FANFT) induced urothelial tumors were treated with purpurin derivatives and red light (> 590 nm, 360 joules/cm2). Treated and control tumors were harvested 12 days after treatment and dry weights compared. At doses of 5.0 ug/g and 2.5 ug/g body weight, the purpurins designated NTl and NT2, when combined with light, caused statistically significant (p < 0.02) tumor regression when compared to light shielded controls. At 1.0 ug/g body weight, NTl and light also induced significant tumor regression (p < 0.02). In previously reported work using the same tumor model, hematoporphyrin derivative (HpD), a widely used tumor photosensitizer, was ineffective in causing tumor regression in doses less than 10 ug/g body weight. Purpurins, which have strong absorption bands above 650 nm and can be synthesized with a high degree of purity, appear to have potential as new photosensit i zers for photodynami c therap_y.
IN VITRO SENSITIVITY OF THREE BLADDER CARCINOMA CELL LINES TO DIFLOUROMETHYLORNITHINE AND METHYLGLYOXAL BIS-GUANYLHYDRAZONE. MUHAMMAD A. BUT,RTJL, NRW YORK, NY (PRESENTATION TO BE MADE BY DR. BULBUL.) Depletion of cellular polyamines by inhibiting biosynthesis has been demonstrated to slow growth of implanted tumors in the experimental animal. Here we report on the effect of ~-diflouromethylornithine (DFMO) an irreversible inhibitor of ornithine decaboxylase and methylglyoxal bisguanylhydrazone (MGBG) a spermidine synthesis inhibitor on tbe in vitro growth of three human bladder tumor cell lines, 2. 5x101l cells of RT4, T24 and 253J human bladder tumor lines were plated seperately on 12 well/plates with RPMI 1640+ 10% fet.al bovine serum. Wells were divided into 4 groups with group 1 serving as control. At day one cells in group 2 were exposed to l.OmM DFMO for 24 hours., cells in group 3 were exposed to 0.04mM MGBG for one hour while cells in group 4 were exposed to DFMO and MGBG sequentially. Cellular proliferation was assessed serially every two days in all groups using (3H) thymidine incorporation assay. Simultanously the levels of cellular polyamines (putrescine, spermidine and spermine) in 4 groups were determined using High Pressure Liquid Chromatography (HPLC). Twenty four hours following the addition of the drugs there was 15%, 30% and 60% inhibition of growth in groups 2,3 and 4 respectively as compared to the control group. After day 3, growth in group 4 remained significantly ()70%) and persistently inhibited while cellular proliferation in groups 2 and 3 recovered and approached that of the control by day seven. These results were consistent for all 3 cell lines with minimal variations. Interestingly the levels of putrescine, spermidine and spermine in the surviving cells, in all groups and time intervals were similar and not significantly different.
126A