Effect of hyperthermia on the action of cis-diamminedichloroplatinum(II), rhodamine 123(2) [tetrachloroplatinum(II)], rhodamine 123, and potassium tetrachloroplatinate in vitro and in vivo

Platinum rhodamine 123 [Pt(Rh-123)2] was synthesized in an effort to produce a new drug which would have the selective uptake into carcinoma cells of Rh-123 and the alkylating and radiosensitizing properties of the chloroplatinum moiety. Because both Rh-123 and cis-diamminedichloroplatinum(II) (CDDP) have been shown to become more cytotoxic at elevated temperatures, we tested the interactions between Pt(Rh-123)2 and hyperthermia both in EMT6 cells in vitro and in the Lewis lung carcinoma in vivo. In the EMT6 cells, CDDP was far more cytotoxic than Pt(Rh-123)2 at 37 degrees C, but its cytotoxicity was less enhanced by exposure of cells to the drug at 42 degrees C than was true for Pt(Rh-123)2 [about 2 logs of increased killing at 42 degrees C after exposure to 10 microM CDDP versus over 3 logs of increased killing at 42 degrees C after exposure to 500 microM Pt(Rh-123)2]. Both Rh-123 and K2PtCl4 also are more cytotoxic to EMT6 cells at 42 degrees C than at 37 degrees C, but the hyperthermic enhancement was far less striking. In the Lewis lung carcinoma, the growth delay produced by CDDP (8 mg/kg) increased by a factor of approximately 2.5 when the drug was given i.p. just prior to local heating of the s.c. thigh tumor to 43 degrees C for 30 min, but the growth delay produced by Pt(Rh-123)2 (100 mg/kg) given i.p. 1 h before local hyperthermia increased by a factor of 5. In contrast, K2PtCl4 and Rh-123 given i.p. produced very short growth delays at normal temperatures and these growth delays were not enhanced by hyperthermia. The effect of these drugs at 37 degrees C and 42 degrees C on the conformation of superhelical pBR322 DNA was also examined. Exposure to CDDP caused progressive alteration from the supercoiled to the linear form of the DNA over time. In contrast, Pt(Rh-123)2 apparently produced progressive degradation of the DNA. Hyperthermia did not alter the qualitative damage produced by the drugs but increased the rate at which the changes occurred. These results suggest both that Pt(Rh-123)2 probably has a different mechanism of action at the DNA than does CDDP and that Pt(Rh-123)2 may be a good drug to use with local hyperthermia and radiation.     

Characterization of a human squamous carcinoma cell line resistant to cis-diamminedichloroplatinum(II)

We have developed a human head and neck squamous cell carcinoma cell line (SCC-25/CP) which is relatively stably resistant to cis-diamminedichloroplatinum(II) (CDDP) after repeated exposure to escalating doses of the drug. The studies reported elucidate the mechanism(s) by which the SCC-25/CP cell line is resistant to CDDP. The SCC-25/CP cell line is approximately 30-fold resistant to CDDP, approximately 10-fold resistant to carboplatin, and about 9-fold resistant to iproplatin. Using [195mPt]CDDP, we examined the levels of platinum in whole cells and cellular fractions of both the SCC-25 and SCC-25/CP cells after 1 h exposure to 100 microM drug. The SCC-25 cells took up 30 pmol of platinum/10(6) cells in 1 h; 64% of the drug was in the nucleus and 21% in the cytosol. The SCC-25/CP cells took up 7 pmol of platinum/10(6) cells; of this, 41% was in the nucleus and 33% in the cytosol. The SCC-25 cell nuclei contained 331 pmol of platinum/mg protein and the cytosol 21 pmol of platinum/mg protein, whereas the SCC-25/CP cell nuclei contained 47 pmol of platinum/mg protein and the cytosol 8.1 pmol/mg protein. The release of drug from both cell lines followed a very similar course and was most rapid over the first 6 h. There was no difference in the non-protein sulfhydryl content of the cell lines. The protein sulfhydryl content, as measured by Ellman's procedure, indicated that the SCC-25/CP cell line has approximately a 2-fold increase in protein sulfhydryl content compared to the SCC-25 cell line. The SCC-25/CP cell line is about 2-fold resistant to cadmium chloride at 50% cell kill and about 2.5-fold resistant at 1 log kill compared to the SCC-25 cell line. Glutathione transferase activity in crude cytoplasmic extracts was measured and found to be approximately 2- to 3-fold higher in the CDDP resistant cells. The isoelectric point of the glutathione transferase isozyme was 4.8 in both the sensitive and resistant cell lines, suggesting induction of the predominant isozyme present in the parent cell line. By alkaline elution there was greater cross-link formation by CDDP in the SCC-25 cell line than in the SCC-25/CP cell line at the same drug concentrations. In conclusion, the mechanism of resistance of the SCC-25/CP cell line to CDDP is multifactorial, involving plasma membrane changes, increased cytosolic binding, and decreased DNA cross-linking.     

Loss of cis-diamminedichloroplatinum(II) resistance in human ovarian carcinoma cells selected for rhodamine 123 resistance.

Cisplatin (DDP)-resistant 2008 human ovarian carcinoma cells (C13*) have an elevated mitochondrial membrane potential that confers hypersensitivity to lipophilic cations. We have addressed whether this change was directly linked to the DDP-resistant phenotype or was a random alteration, unrelated to resistance. The elevated mitochondrial membrane potential and ensuing rhodamine 123 (Rh123) hypersensitivity of C13* cells provided a positive selection rationale. Cells with low mitochondrial membrane potential will have a survival advantage over those with a high mitochondrial membrane potential, when exposed to Rh123. We therefore used Rh123 to isolate revertants (RH4) from the 12-fold DDP-resistant C13* cell line that had a low mitochondrial membrane potential. RH4 cells had parental (2008) mitochondrial membrane potential levels as shown by flow cytometry analysis of Rh123 stained cells and by tetraphenylphosphonium cation uptake. The RH4 cells lost a substantial portion of their DDP resistance such that they were only 2- to 3-fold resistant to DDP. Despite this major loss of resistance, they retained a number of the phenotypic changes related to DDP resistance, observed in C13* cells. RH4 cells displayed the same DDP accumulation defect, 2-fold elevated glutathione levels and 2-fold resistance to CdCl2 as C13* cells. We conclude that although the biochemical mechanism by which an elevated mitochondrial membrane potential elicits DDP resistance is unknown, these mitochondrial changes are central to the expression of DDP resistance in C13* cells.     

Mitochondrial defects in cis-diamminedichloroplatinum(II)-resistant human ovarian carcinoma cells.

We have been studying the membranes of cisplatin (DDP)-resistant 2008 human ovarian carcinoma cells (C13* cells) for alterations that may account for their decreased DDP accumulation. We now report that C13* cells have significant changes in their mitochondrial and plasma membrane potentials and in their mitochondrial morphology. C13* cells accumulated 2.0 +/- 0.1-fold more of the membrane potential marker [3H]tetraphenylphosphonium cation (TPP+) than sensitive cells. In high K+ medium, which depolarizes the plasma membrane but not the mitochondrial membrane, [3H]TPP+ accumulation was still 2.3 +/- 0.1- fold greater in resistant cells, indicating that the mitochondrial membrane potential was higher. In the presence of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, which depolarizes the mitochondrial membrane but not the plasma membrane, [3H]TPP+ accumulation demonstrated that the plasma membrane potential in C13* cells was elevated as well. These elevations were also present in C8 cells with low-level DDP resistance. After ouabain treatment, exposure to nigericin stimulated [3H]TPP+ accumulation 3-fold in sensitive cells but had no effect in C13* cells, indicating either that: (a) the mitochondrial pH gradient was minimal; or (b) the mitochondrial electric potential was already at a maximal level in C13* cells. Fluorescence microscopy of living cells stained with the mitochondria-specific dye rhodamine 123 revealed that resistant cells had significant changes in their mitochondrial morphology. Electron microscopy also revealed major alterations in the cristae structure. The C13* cells, which were approximately 15-fold resistant to DDP, were 5-fold hypersensitive to the mitochondrial poison rhodamine 123. We conclude that these DDP-resistant 2008 cells have an elevated plasma membrane potential and alterations in their mitochondria as indicated by their membrane potential, morphology, and sensitivity to mitochondrial poisons. These results imply that mitochondria play an important role in the cellular pharmacology of DDP.     

Effect of hyperthermia on rhodamine 123 cytotoxicity in doxorubicin-sensitive and doxorubicin-resistant human breast carcinoma cell lines in vitro

Rhodamine 123 (Rh123) cytotoxicity and intracellular accumulation were studied in normothermic and hyperthermic conditions in a human breast carcinoma cell line (MCF7/WT) and its doxorubicin-resistant subline (MCF7/Dox^R). MCF7/Dox^R cells were resistant to hyperthermia and Rh123. Hyperthermic potentiation of Rh123 cytotoxicity was present in MCF7/WT but not in MCF7/Dox^R cells. Results suggest that the effect observed in MCF7/WT cells is related to a heat-induced increased accumulation of Rh123 and not to a heat-induced activation of the drug. A low basal uptake and a fast release of Rh123 could explain the resistance of MCF7/Dox^R to the drug. Resistance to Rh123 and lack of a heat-induced increased uptake account for the lack of hyperthermic enhancement of Rh123 cytotoxicity in the resistant cells.     

Characterization of a cis-diamminedichloroplatinum(II)-resistant human ovarian cancer cell line and its use in evaluation of platinum analogues

Human ovarian cancer cell lines with stable cisplatin resistance have been developed by chronic exposure of the parent cisplatin-sensitive A2780 line to increasing concentrations of cisplatin. 2780CP8 (CP8 refers to this cell line's growth in medium containing 8 microM cisplatin) has several clonal cytogenetic abnormalities but lacks homogeneously staining regions or double-minute chromosomes. It has a significantly greater monolayer growth rate, cloning efficiency in agarose, and total glutathione content compared to the A2780 line, but similar activities of several glutathione-dependent enzymes. The 2780CP8 subline is 7.3-fold resistant to cisplatin compared to the A2780 line, as well as cross-resistant to irradiation and melphalan. It is not cross-resistant to Adriamycin, but this develops with increased cisplatin resistance (14-fold) obtained by further cisplatin exposure of 2780CP8. Of the cisplatin analogues tested which are of current clinical interest, carboplatin, iproplatin, and tetraplatin, only the latter is more cytotoxic than cisplatin in the A2780 and 2780CP8 lines. The 2780CP8 subline is also cross-resistant to these analogues in the relative order carboplatin greater than iproplatin greater than tetraplatin (most to least cross-resistant). Treatment of a highly cisplatin resistant cell line (2780CP70) with either melphalan or cisplatin was associated with a significant increase in [3H]thymidine incorporation into DNA in the presence of 10 mM hydroxyurea compared with the parent sensitive cell line which showed essentially no capacity to repair DNA damage by these drugs. A2780 and its cisplatin-resistant cell lines may thus be useful in studying drug resistance mechanisms, in screening new drugs for activity (especially against drug resistant tumors), and in formulating induction and salvage therapies for ovarian cancer.     

Mechanisms of collateral sensitivity to fluorouracil of a cis-diamminedichloroplatinum(II)-resistant human non-small lung cancer cell line.

A cisplatin(CDDP)-resistant subline of a human lung cancer cell line, PC-7/CDDP, was 4.7-fold more resistant to CDDP than the parent line in a colony-forming assay. The sensitivity of this cell line to anthracyclines, vinca-alkaloid, etoposide, mitomycin C, and bleomycin was similar to that of the parental line, PC-7. However, PC-7/CDDP exhibited 4-fold higher sensitivity to fluorouracil (FUra). Possible mechanisms associated with the collateral sensitivity to FUra were studied in PC-7/CDDP cells. The sensitivity of both cell lines to FUra did not correlate with the effect of FUra on RNA. On the other hand, FUra induced a greater reduction in dTTP pools and more single strand breaks in PC-7/CDDP than in PC-7 cells. These results suggest that the pathway for de novo deoxyribonucleotide synthesis may be a target for FUra in PC-7/CDDP cells. However, inhibition of thymidylate synthase after FUra treatment did not correlate with the DNA-directed activity of FUra. Based on the above findings, the decreased salvage synthesis of dTTP was considered a possible mechanism of the greater reduction of dTTP pools in PC-7/CDDP cells. However, the activity of dThd kinase was the same in both cell lines. In the presence of physiological concentrations of exogenous dThd in the serum, uptake of dThd was less in PC-7/CDDP cells than that in PC-7 cells. Our data suggest that FUra-induced cytotoxicity in PC-7/CDDP cells is associated with the inhibition of dTTP synthesis and that the decreased uptake of dThd is a possible mechanism of the collateral sensitivity to FUra in PC-7/CDDP cells.     

cis-diamminedichloroplatinum(II)-resistant sublines derived from two human ovarian tumor cell lines

In two human ovarian tumor cell lines, resistance to cis-diamminedichloroplatinum(II) (CDDP) was induced by continuous exposure of the parental lines to an increasing CDDP concentration in the culture medium. In contrast, a six times repeated pulse exposure of 6.7 microM or 16.7 microM CDDP for 1 h did not result in a cell line that showed a higher survival in CDDP-containing medium. The ID50 value for CDDP was seven to eight times higher for the resistant lines and those lines were able to grow at 3.3 microM CDDP. The induced resistance was stable during at least 25 doubling times. The CDDP-resistant sublines showed cross-resistance to the CDDP-analogues cis-diammine-1,1-cyclobutane-dicarboxylateplatinum(II) and cis-dichlorobis(isopropylamine)-trans-dihydroxyplatinum(IV) indicating that resistance to the different platinum compounds is generated by a common mechanism. The resistant sublines were also cross-resistant to mitomycin C and melphalan. The degree of cross-resistance for the tested drugs varied widely between the two cell lines. Resistance to CDDP was clearly correlated to decreased amounts of platinum in the resistant cells as compared to the sensitive cells. The amplification and expression of genes encoding proteins that had been shown to be involved in multidrug resistance, e.g., the Mr 170,000 P-glycoprotein was also studied. No amplification or overexpression of these genes could be shown in the resistant cell lines.     

Single step selection of cis-diamminedichloroplatinum(II) resistant mutants from a human ovarian carcinoma cell line

We have shown that cis diamminedichloroplatinum-(II) (DDP) resistant mutants can be isolated from the human ovarian carcinoma cell line A2780 using a single-step selection protocol with DDP. DDP resistant colonies were calculated to be present at a frequency of 1.7 x 10(-6)/viable cell using a fluctuation analysis. The mutational origin of these surviving colonies is inferred by the fact that their frequency is increased by treatment of the A2780 cells with the chemical mutagen ethyl methane-sulfonate, with a maximum frequency observed after a 3-day expression time. Independently isolated clones maintain, in the absence of selection, a DDP resistant phenotype up to 7-fold more resistant than the parental A2780 cells. The resistance modifiers aphidicolin and buthionine sulfoximine have no effect on the frequency of DDP resistant mutants. Therefore neither of these drugs appears to have an effect on increasing the sensitivity of DDP resistant mutants existing in a cell population prior to DDP exposure.     

Mechanisms of resistance to cis-diamminedichloroplatinum (II) in a rat ovarian carcinoma cell line

A cis-diamminedichloroplatinum(II) (cisplatin)-resistant subline (Cis-Pt^r) demonstrated 20-fold greater resistance to the cytotoxic effects of cisplatin, compared with the parental cloned rat ovarian carcinoma cell line (ROT 68/C1). The uptake of cisplatin into the Cis-Pt^r cells was identical to that into the ROT68/C1 cells in vitro and in vivo. Glutathione activity in a cytoplasmic extract was 1.4-fold and 1.8-fold greater in the Cis-PT^r cells than in the ROT68/C1 cells in vitro and in vivo, respectively. There was no difference between the ROT68/C1 and Cis-PT^r cells in ^195mcisplatin binding per μg DNA. DNA repair of cisplatin DNA damage was increased in the Cis-PT^r cells but not in the ROT68/C1 cells. These results suggest that the mechanism of resistance to cisplatin in rat ovarian carcinoma cells involve increased activity of the DNA repair system and increased cytosolic binding to thiols may also be involved.     

Characterization of a human small cell lung carcinoma cell line with acquired resistance to cis-diamminedichloroplatinum(II) in vitro

A 6.4-fold cis-diamminedichloroplatinum(II) (CDDP) resistant human small cell lung carcinoma cell line (GLC4-CDDP) was developed to study acquired CDDP resistance in vitro. Compared to the sensitive cell line (GLC4), the GLC4-CDDP showed an increase in doubling time and a decrease in cloning efficiency, cellular size, double minutes per cell, cellular protein, and nuclear protein content. While a complete cross-resistance for tetraplatin and a partial cross-resistance for doxorubicin, melphalan, cadmium chloride, carboplatin, and cis-dichloro-trans-dihydroxo-cis-bis(isoprolylamine)platinum (IV) (resistance factor, respectively,4.0,5.8,2.1,1.5,2.9) was found, no cross-resistance for vincristine was found. In the GLC4-CDDP line in comparison to the GLC4 line, glutathione and total amount of sulfhydryl compounds was significantly increased, while glutathione S-transferase and glutathione reductase was the same. The platinum content in cells and nuclei was lower in the resistant line, but after correction for cellular protein or volume no difference was found. The amount of platinum bound to DNA was significantly lower in the GLC4-CDDP line. After a 1-h incubation with CDDP, the amount of Pt-GG adducts was the same and the amount of interstrand cross-links was reduced in the GLC4-CDDP line as compared to GLC4. In conclusion, in the GLC4-CDDP line the phenotype and genotype are changed and various mechanisms, such as decreased Pt-DNA binding, elevated glutathione, and reduced interstrand cross-links, play a role in the development of the CDDP resistance.     

Inhibition by 5-fluorouracil of cis-diamminedichloroplatinum(II)-induced DNA interstrand cross-link removal in a HST-1 human squamous carcinoma cell line.

To elucidate the mechanism of the synergistic cytotoxicity of 5-fluorouracil (5-FU) and cis-diamminedichloroplatinum(II) (CDDP), we studied the interaction of these agents using a human squamous carcinoma cell line (HST-1). Exposure to 5-FU for 24 h and to CDDP for 1 h produced a 50% inhibitory concentration of 1.0 micrograms/ml (7.7 microM) and 2.5 micrograms/ml (8.3 microM), respectively. The cytotoxic action of CDDP was augmented, and a greater than additive effect was observed when the cells were exposed to 5-FU (1.0 micrograms/ml; 7.7 microM) for 24 h before the CDDP treatment. This synergistic activity was maximal when the interval between 5-FU and CDDP exceeded 24 h. In contrast, the cytotoxicity of CDDP was attenuated when it preceded the exposure to 5-FU. Thymidine did not alter the 5-FU-CDDP interaction. Evaluation of the kinetics of the removal of DNA interstrand cross-links, measured by alkaline elution, showed a significant reduction of this removal in the cells exposed to 5-FU followed by CDDP with a drug-free interval of 48 h, as compared with cells exposed to CDDP alone, or to 5-FU immediately followed by CDDP, although no differences were found in the formation of DNA interstrand cross-links by CDDP among these cells. No significant differences in the accumulation of intracellular platinum were detected by atomic absorption spectrophotometry. These findings suggest that 5-FU modulates the repair of platinum-DNA adducts, thereby potentiating the antitumor activity of CDDP.     

Distinct P-glycoprotein expression in two subclones simultaneously selected from a human colon carcinoma cell line by cis-diamminedichloroplatinum (II)

Two drug-resistant sublines, CP2.0 and RT, were simultaneously selected by cis-diamminedichloroplatinum (CDDP) from the human colon carcinoma cell line LoVo by the conventional method of continuous drug exposure. The 2 sublines differed in morphology, growth kinetics and pattern of gene expression. Genetic signature analysis indicated that the lines were independent subclones but that both arose from LoVo. These sublines were maintained in a growth medium containing 2.0 μg/ml CDDP. However, CP2.0 cells were 3 times more resistant to CDDP than were RT cells. Although both were cross-resistant to mustargen and 5-fluorouracil, only CP2.0 was resistant to Adriamycin and vincristine. Western-blot analysis, immunocytochemical staining and in vitro phosphorylation experiments indicated that the level of P-glycoprotein was significantly elevated in CP2.0 but not in RT. Despite the differences between these sublines, they possess similar CDDP-resistance mechanisms, including decreased intracellular CDDP accumulation, elevated levels of glutathione and metallothionein-like proteins, increased glutathione transferase-μ mRNA, and enhanced susceptibility to CDDP cytotoxicity after treatment with DL-buthionine-[S,R]-sulfoximine. Nevertheless, our results suggest that, in certain tumor types, P-glycoproteinmediated multi-drug resistance and CDDP-resistance phenotypes can coexist in cells with primary resistance to CDDP.     

Interaction with hyperthermia of tetrachloroplatinum(II)(Nile blue)2 and tetrachloroplatinum(II)(neutral red)2 in EMT6 murine cells and the murine FSaIIC fibrosarcoma

Complexes of the tetrachoroplatinum(II) dianion with positively charged nuclear dyes were prepared in an effort to produce agents which gain ready access into the nucleus and become very cytotoxic at clinically relevant hyperthermia temperatures. Pt(Nile blue)2 and Pt(neutral red)2 are complexes of tetrachloroplatinum(II) with two closely related p-quinonediamine dyes. Pt(Nile blue)2 and Pt(neutral red)2 were only moderately cytotoxic to exponentially growing normally oxygenated or hypoxic EMT6 cells in vitro at pH 7.40 and 37 degrees C. At pH 7.40 and 42 degrees C and especially at 43 degrees C, however, Pt(Nile blue)2 became far more cytotoxic. At pH 6.45 Pt(Nile blue)2 became more toxic toward hypoxic cells (cell kill of 3.5 logs at 500 microM, 42 degrees C for 1 h). Pt(neutral red)2 became much more cytotoxic at pH 6.45 and 42 degrees C or 43 degrees C compared to pH 7.4, and the cell kill observed was similar in both euoxic and hypoxic cells (3 logs at pH 6.45, 43 degrees C with only 100 microM). Tumor cell survival studies in the FSaIIC murine fibrosarcoma demonstrated that both drugs killed in a dose-dependent log-linear manner. Hyperthermia treatment (43 degrees C, 30 min) immediately after either drug resulted in a dose modifying effect. The tumor growth delay produced by Pt(Nile blue)2 (100 mg/kg) was 4.6 days and by Pt(neutral red)2 (100 mg/kg) was 3.8 days. Both drugs were markedly improved by hyperthermia (tumor growth delay 1.4 days for hyperthermia; tumor growth delay 10.9 days for Pt(Nile blue)2 and 8.0 days for Pt(neutral red)2. Intracellular platinum levels were approximately 200 times higher after exposure of EMT6 cells to 25 microM of Pt(Nile blue)2 or Pt(neutral red)2 for 1 h at 37 degrees C than after exposure to the same concentration of cis-diamminedichloroplatinum(II). Treatment of cells with the drugs at 42 degrees C (1 h) resulted in no change in platinum levels with cis-diamminedichloroplatinum(II), but with Pt(Nile blue)2 and Pt(neutral red)2 an increase of 2- to 3-fold was found. Since previous work has shown that both of these complexes are active radiosensitizing agents, these new drugs seem quite well suited for further development as antitumor agents for use against solid tumors alone and in conjunction with hyperthermia and/or radiation therapy.     

Alphoid DNA nucleotide sequences from two human cell lines and the corresponding cis-diamminedichloroplatinum(II)-resistant sublines

In order to study the interaction between cis-diamminedichloroplatinum(II) (CDDP) and representative human DNA in a highly defined manner, alphoid sequence DNA was isolated from two human parental cancer lines (one of head and neck squamous cell origin, SCC-25, and one of breast carcinoma origin, MCF-7) as well as from three CDDP-resistant cell lines derived from the parental lines. The alphoid DNAs were then cloned and tested for homology with published consensus sequence results. Percent homology with the consensus sequence varied between 84.4% and 91.7% for all of the cloned alphoid DNA tested and there was no significant difference for parentally derived versus resistant subline derived alphoid DNA. These results suggest, as expected, that resistance to the mutagenic chemotherapeutic drug CDDP is not the result of a general alteration in DNA base sequence from guanine and adenine to cytosine and thymidine, which are less favorable binding sites. The highly defined, abundant alphoid sequence DNA should provide an excellent model for investigating the interaction between various DNA active drugs and human DNA.     

Effect of cis-diamminedichloroplatinum(II) combined with whole body hyperthermia on renal injury

The effect of whole body hyperthermia (WBH) on cis-diamminedichloroplatinum (II) (DDP) induced renal toxicity and antitumor effect was studied using a F344 rat model. Renal injury at 5 and 14 days after treatment was evaluated using animal mortality, renal functional assays (blood urea nitrogen, creatinine), and histopathological methods. WBH (120 min at 41.5 degrees C) enhanced both antitumor effects and toxic side effects. The latter included increased mortality, increased blood urea nitrogen and creatinine levels, and increased renal damage. After simultaneous treatment with WBH and DDP, thermal enhancement ratios (TER) for renal damage between 2.5 and 3.0 were calculated. The histopathological changes observed in the kidney after DDP alone or combined with WBH were primarily found in the proximal pars recta tubules (S3 segment) in the outer stripe of the outer medulla. There was no qualitative difference in tubular damage between rats treated with DDP alone or those treated with DDP combined with WBH. However, at a fixed DDP dose, damage in the combined treatment modality group was significantly greater than in the DDP-only treated group.     

Increased removal of DNA-bound platinum in a human ovarian cancer cell line resistant to cis-diamminedichloroplatinum(II)

A human ovarian cancer cell line resistant to cis-diamminedichloroplatinum(II) (DDP) (2780CP) was compared with its DDP-sensitive parental cell line (A2780) to determine whether differences in the removal rate of DNA-bound platinum were related to resistance. Both cell lines were treated in vitro with various doses of DDP for 2 h and subsequently incubated in arginine-deficient Eagle's minimum essential medium with 2.5% dialyzed fetal bovine serum in the presence or absence of aphidicolin. After 0, 12, and 24 h, DNA was isolated from the cells and DNA-bound platinum was determined by flameless atomic absorption spectrophotometry. Binding of platinum to DNA of either cell line was a linear function of concentration ranging from 20 to 80 microM DDP. Platinum binding was almost equal at each dose in both cell lines. 2780CP cells that were 3-fold resistant to DDP lost 30.5 to 40.1% of their total DNA-bound platinum, compared with a 1.3 to 16.1% loss for A2780 cells, 12 to 24 h after a 2-h exposure to 40 microM DDP, respectively. Aphidicolin (3.0 micrograms/ml) increased the cytotoxicity in 2780CP cells by about 2-fold and caused a significant delay in the time required for platinum removal in the resistant cells (14.6 and 18.9% at 12 and 24 h). These studies indicate that the mechanism of DDP resistance in the 2780CP cell line is related to an increased ability to remove platinum-DNA adducts, and not to a difference in initial DDP binding to DNA.     

热疗联合紫杉醇对人舌鳞癌细胞系CAL-27增殖、凋亡和周期影响

目的:观察热疗联合紫杉醇对人舌鳞癌细胞系CAL-27增殖、凋亡和周期的影响并探讨其机制。方法:CCK-8法确定紫杉醇工作浓度,将体外培养CAL-27细胞分为对照组、紫杉醇组、42℃热疗组和联合治疗组。克隆形成实验检测细胞增殖能力,流式细胞术检测细胞周期及凋亡,蛋白印迹法检测AKT、p-AKT、Bcl-2、Bax蛋白表达...     

N1,N12-bis(ethyl)spermine effect on growth of cis-diamminedichloroplatinum(II)-sensitive and -resistant human ovarian-carcinoma cell lines

The results presented here demonstrate that a cis-diamminedichloroplatinum(II) (DDP)-resistant human ovarian-carcinoma cell line is also cross-resistant to the spermine analogue N¹,N¹²-bis(ethyl)spermine (BESPM). We report that C13* cells, which are approximately 20-fold resistant to DDP, similarly showed 7-fold resistance to BESPM by colony-forming assay with an IC₅₀ value of 24.6 ± 2 μM vs. 3.4 ± 0.8 μM of 2008 cells. Resistance appears to be the result of many effects, such as different morphological and functional modifications of mitochondria. Furthermore, although BESPM accumulation was almost identical in sensitive and resistant cells, the intracellular polyamine pool of the 2 cell lines was differentially affected by this polyamine analogue. In fact, when spermidine (SPD) was still detectable in C13* cells, in 2008 cells it was not, and the spermine (SPM) content was always more markedly reduced in sensitive cells than in the resistant variant. The lower polyamine content of 2008 cells could be related to a higher degree of induction of spermidine/spermine N¹-acetyltransferase (SSAT) activity by BESPM in sensitive cells than in their resistant counterpart. Despite the observed cross-resistance, the combination of the 2 drugs resulted in supra-additive and synergistic effects in both cell lines, depending on concentration, as assessed by median-effect analysis of the survival data. The effectiveness of this combination was also confirmed by the increased accumulation of cells in the G₂/M phase of the cell cycle in both cell lines. Taken together, these data suggest that BESPM effect on cell growth of DDP-sensitive and DDP-resistant cells involves multiple mechanisms that are differently modulated by the DDP-resistant phenotype. Int. J. Cancer 78:33–40, 1998.© 1998 Wiley-Liss, Inc.     

X-ray and cis-diamminedichloroplatinum(II) cross-resistance in human tumor cell lines

Clinical studies have suggested a close correlation between cis-diamminedichloroplatinum(II) (cisplatin) and radiation resistance. To determine whether this cross-resistance is due to an inherent cellular resistance to both agents, ten early passage human tumor cell lines were examined for their radiation and cisplatin sensitivity in vitro. Previous studies have suggested that these early passage tumor cell lines retain many of their in vivo characteristics and are therefore good models for tumor cells in vivo. Radioresistance was strongly associated with cisplatin resistance in these cell lines. Four of the cell lines examined were radioresistant, having Dos greater than 2.0 Gy. These four lines were also resistant to cisplatin, with the dose reducing survival to 10% greater than 1.29 microM. The remaining six cell lines had Dos ranging from 1.07 to 1.57 Gy of X-ray and doses reducing survival to 10% of less than 0.83 microM cisplatin. Because early passage human tumor cell lines were used, resistance or sensitivity to radiation and cisplatin most likely developed in vivo and was not due to selection in vitro. These results indicate that cross-resistance between cisplatin and radiation in vivo is probably due primarily to an inherent cellular resistance to these agents and not necessarily to the tumor microenvironment in situ.