4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Adenocarcinoma

We used a 20-mer antisense oligonucleotide against human MDR1 mRNA to study the mechanism of uptake of oligonucleotides by a human colorectal adenocarcinoma cell line, HCT-15.5'-end-32P-labeled oligonucleotides were mainly used. The uptake of phosphodiester oligonucleotides (D-oligo) by HCT-15 cells increased with time, but the uptake of phosphorothioate oligonucleotides (S-oligo) showed minimal increases with time. HeLa cells showed a 4-fold greater D-oligo uptake than did HCT-15 cells, and other cell lines exhibited a 25-fold lower uptake than did HCT-15 cells. On the other hand, S-oligo uptake varied only severalfold among the cell lines used. Doligo up-take consisted of a saturable component (Michaelis constant K(m) = 0.16 microM and maximal uptake rate Vmax = 4.8 pmol/min/mg for HCT-15 cells and K(m) = 0.21 microM and Vmax = 35.6 pmol/min/mg for HeLa cells), i.e., the difference between the cell types was mainly in Vmax. The uptake of labeled D-oligo was inhibited by unlabeled S-oligo at a much lower concentration (Ki = 0.01 microM) than by unlabeled D-oligo (K(m) = 0.16 microM), although the uptake rate of D-oligo was greater than that of S-oligo. The D-oligo uptake was highly temperature dependent. Of the sulfhydryl-modifying reagents (p-chloromercuriphenyl-sulfonic acid and p-chloromercuribenzoic acid), only p-chloromercuribenzoic acid inhibited D-oligo uptake, suggesting the involvement of a protein whose sulfhydryl residues inside the cell are critical for D-oligo uptake. Chloroquine and monensin, which are known to alter the intracellular fate of ligands and receptors, did not affect D-oligo uptake, but phenylarsine oxide, an inhibitor of internalization processes, inhibited uptake significantly. Down-regulation and recovery of D-oligo uptake induced by excess unlabeled D-oligo pretreatment occurred, but this was only partial. Studies with a digitonin equilibrium method and confocal microscopy indicated that intracellular localization of D-oligo was mainly in a vesicular compartment. More stable 3'-end-32P-labeled oligonucleotides were also used, to compare their uptake characteristics with those of the 5'-end-labeled oligonucleotides. The uptake characteristics for the two labels were similar in terms of saturability, greater D-oligo uptake, compared with S-oligo, and the inhibitory effect of p-chloromercuribenzoic acid; however, the stable 3'-end label showed greater uptake than the 5'-end label. Our findings suggest that D-oligo uptake by HC

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Adenocarcinoma; Anions; Cell Compartmentation; Colorectal Neoplasms; Down-Regulation; Endocytosis; Genes, MDR; Humans; Ion Channels; Kinetics; Microscopy, Confocal; Oligonucleotides, Antisense; Structure-Activity Relationship; Temperature; Time Factors; Tumor Cells, Cultured

Intracellular pH is regulated mainly by Na+/H+ antiport and Cl-/HCO3- exchange through the cell membrane. Amiloride (3,5-diamino-6-chloro-N-(diaminomethylene)pyrazine carboxamide) is a diuretic drug that blocks Na+/H+ antiport and DIDS (4,4-diisothiocyanatostilbene-2,2'-disulfonic acid) is an inhibitor of Cl-/HCO3- exchange. We investigated the potency of these drugs to lower pHi and increase the thermosensitivity of tumors in vivo.. The cytocidal effect of heat in combination with drug effect in vivo was studied using the in vivo-in vitro clonogenic assay method and the tumor growth delay method with SCK tumors, a mammary adenocarcinoma, on the hind limbs of A/J mice. The effects of amiloride and DIDS on tumor pHi and high energy phosphate levels were investigated using 31P-NMR.. We observed that amiloride or DIDS alone increased the effect of hyperthermia at 42.5 degrees C or 43.5 degrees C to suppress tumor growth. The thermosensitization was greater when the two drugs were combined. For example, hyperthermia at 43.5 degrees C alone resulted in a tumor growth delay of about 4 days. When 10 mg/kg amiloride or 25 mg/kg DIDS was injected prior to heating, the growth delay increased to about 6 days. When both drugs were injected prior to heating, a total growth delay of 8 days was obtained. In vivo-in vitro excision assays for cell survival demonstrated that these drugs enhanced the heat-induced tumor cell death. An i.p. injection of 10 mg/kg amiloride plus 25 mg/kg DIDS did not lower the tumor pHi over a 120 min interval. Heating the tumors at 42.5 degrees C for 1 hr significantly lowered the pHi and when the tumor-bearing mice were injected i.p with amiloride and DIDS, and the tumors were heated 1 hr later, the drop in pHi was greater relative to that by heating alone. Heating alone significantly lowered the tumor energy levels as indicated by PCr/Pi and beta-ATP/Pi ratios and an i.p. injection of 25 mg/kg amiloride prior to heating further reduced the energy status in the tumors.. Amiloride or its analogs and DIDS may be useful in increasing the therapeutic efficacy of hyperthermia treatments by enhancing the reduction in tumor pHi.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenocarcinoma; Amiloride; Animals; Cell Survival; Combined Modality Therapy; Drug Combinations; Female; Hyperthermia, Induced; Mammary Neoplasms, Experimental; Mice; Neoplasm Transplantation

The effects of amiloride (an inhibitor of Na+/H+ antiport), DIDS (an inhibitor of Na(+)-coupled and Na(+)-independent HCO3-/Cl- exchange) and nigericin (K+/H+ ionophore) alone and in various combinations on the intracellular pH (pHi) and thermosensitivity of SCK tumor cells were studied. Hyperthermia alone at 43 degrees C for 2 h decreased pHi of SCK cells by 0.15-0.20 pH units, as measured fluorometrically using the pH-sensitive dye BCECF. When the cells were treated with 0.5 mM amiloride at 37 degrees C, the pHi declined by 0.10-0.15 pH units at an extracellular pH (pHe) of both 7.2 and 6.6. Amiloride at 0.5 mM enhanced the thermal damage to SCK cells at pHe 6.6 but not at pHe 7.2. DIDS alone at 0.1 mM exerted no effect on pHi or cellular thermosensitivity. DIDS, however, enhanced the effects of amiloride in decreasing pHi and in increasing the thermoresponse of SCK cells, particularly at pHe 6.6. Treatment of the cells with nigericin at 0.1-1.0 micrograms/ml lowered the pHi and enhanced the thermosensitivity of the cells in a dose-dependent manner. Reductions in pHi and increases in thermosensitivity by nigericin at the lower concentration at pHe 6.6 were far greater than at pHe 7.2. When a mixture of 1.0 micrograms/ml nigericin, 0.5 mM amiloride, and 0.1 mM DIDS was present in the medium, the pHi rapidly decreased by about 0.3 and 0.4 pH units at pHe 7.2 and 6.6, respectively. This drug combination was also extremely effective in sensitizing SCK cells to heat, particularly at pHe 6.6. The fact that the thermosensitization by these drugs at pHe 6.6 is more pronounced than at pHe 7.2 and that intratumor environments are known to be acidic strongly suggested that it may then be possible to enhance the thermal damage with such drugs preferentially in tumors relative to normal tissues.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adaptation, Physiological; Adenocarcinoma; Amiloride; Animals; Cell Line; Hot Temperature; Hydrogen-Ion Concentration; In Vitro Techniques; Mammary Neoplasms, Animal; Mice; Nigericin

The factors which influence the exocytosis of mucins are not well characterized. Since the physical properties of mucins may be affected significantly by the co-secretion of electrolytes and water, we studied the relationship between ion movement and mucin secretion in T84 cells, a human colonic adenocarcinoma cell line which has been well characterized with respect to apical chloride secretion. Secretion of mucin was assessed by immunoassay of mucin appearing in the medium within 30 min of stimulation. Cells were grown on plastic in DMEM/Ham's F12 medium and experiments were carried out at 70% confluence. Mucin secretion was stimulated by the calcium ionophore A23187, or A23187 plus vasoactive intestinal polypeptide. Stimulated mucin secretion was not affected by loop diuretics (furosemide (1 x 10(-3) M) or bumetanide (1 x 10(-4) M)), with or without the addition of ouabain (5 x 10(-5) M) and amiloride (1 x 10(-5) M), making it unlikely that transcellular chloride movements in necessary for mucin secretion. However, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; (1 x 10(-5) and 5 x 10(-5) M) and three potassium channel blockers BaCl2 (1 x 10(-3) and 5 x 10(-3) M), tetraethylammonium chloride (1 x 10(-2) M) and quinine (5 x 10(-4) M) inhibited mucin secretion. A DIDS-sensitive chloride channel or chloride/bicarbonate exchanger and a Ca2(+)-dependent potassium channel may play important roles in mucin secretion. Since plasma membranes are sparingly permeable to DIDS, the DIDS-sensitive site is likely to be on the apical plasma membrane, perhaps at an initiation locus for exocytosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenocarcinoma; Amiloride; Barium; Barium Compounds; Calcimycin; Cell Line; Chlorides; Colonic Neoplasms; Furosemide; Humans; Kinetics; Mucins; Ouabain; Potassium Channels; Quinine; Stilbenes; Tetraethylammonium; Tetraethylammonium Compounds; Tumor Cells, Cultured; Vasoactive Intestinal Peptide