Role of reactive oxygen species in cis-dichlorodiammineplatinum-induced cytotoxicity on bladder cancer cells.

This study was undertaken to investigate the intracellular induction of reactive oxygen species (ROS) by cis-dichlorodiammineplatinum (CDDP) and the augmentation of their cytotoxicity in bladder cancer cells (KU7) by enhancement of ROS generation by the glutathione (GSH) depletors buthionine sulphoximine (BSO) and diethylmaleate (DEM). CDDP-induced cytotoxicity in KU7 cells and its modulation by GSH depletors were determined using spectrophotometric measurement with crystal violet staining. The effects of GSH depletors on intracellular GSH levels were confirmed using the GSH reductase-DTNB recycling method. Intracellular ROS generation induced by CDDP with or without GSH depletors was estimated from the amount of intracellular dichlorofluorescein (DCF), an oxidized product of dichlorofluorescein (DCFH), which was measured with an anchored cell analysis and sorting system. The cytotoxic effects of CDDP (IC50 15.0 +/- 2.5 microM) were significantly enhanced by BSO (IC50 9.3 +/- 2.6 microM, P < 0.01) and DEM (IC50 10.3 +/- 0.3 microM, P <0.01). BSO and DEM produced a significant depletion in intracellular GSH levels (9.6 +/- 0.4 nmol 10(-6) cells, 17.9 +/- 1.0 nmol 10(-6) cells) compared with the controls (30.5 +/- 0.6 nmol 10(-6) cells). Intracellular DCF production in KU7 cells treated with CDDP (1.35 +/- 0.33 microM) was significantly enhanced by the addition of BSO (4.43 +/- 0.33 microM) or DEM (3.12 +/- 0.22 microM) at 150 min. These results suggest that ROS may play a substantial role in CDDP-induced cytotoxicity and that GSH depletors augment its cytotoxicity through an enhancement of ROS generation in bladder cancer cells.     

A role of glutathione in resistance mechanisms to cis-diamminedichloroplatinum(ii) in human ovarian-cancer cell-lines in-vitro

The effects of D,L-buthionine-S, R-sulphoximine (BSO) on the cellular glutathione (GSH) levels and cis-diamminedichloroplatinum (II) (CDDP) sensitivity of human ovarian cancer cell lines (HRA, KK and MH) with different sensitivity to CDDP were examined. HRA cells were derived from ascites of a patient with serous cystadenocarcinoma of the ovary who responded well to CDDP-based combination chemotherapy. KK and MH cells were also established from ascites of a patient with clear cell carcinoma of the ovary and with serous cystadenocarcinoma of the ovary, respectively who did not respond to CDDP-based combination chemotherapy. The KK and MH cell lines showed 2.93- and 7.80-fold higher resistance to CDDP in vitro than HRA cells, respectively and had also cross-resistance to its analogues. Although GSH levels in the CDDP sensitive HRA cells were 33.4 nmol/10(7) cells, the CDDP resistant KK and MH cells showed 5.61-and 10.48-fold higher GSH levels, respectively. The higher the GSH levels the more rapid depletion of the cellular GSH by BSO occurred. Cystine, a component of GSH, resulted in an increase of the cellular GSH in these cell lines. When the cellular GSH levels were changed by BSO or cystine, changes of the IC50 to CDDP and its analogues of the CDDP resistant KK and MH cells correlated with those of the cellular GSH levels while the IC50 values of the CDDP sensitive HRA cells remained unchanged. In addition, when these resistant cell lines (but not HRA cells) were incubated with CDDP, the cellular GSH levels markedly increased. From these results, we conclude that GSH may have a role in the mechanisms of intrinsic resistance to CDDP and its analogues of the KK and MH cell lines.     

N-acetylcysteine modifies cis-dichlorodiammineplatinum-induced effects in bladder cancer cells.

We previously demonstrated a role of reactive oxygen species (ROS) in cytotoxicity induced by cis-dichlorodiammineplatinum (CDDP) in combination with glutathione (GSH) depletors in bladder cancer cells. However, the relationship between CDDP and ROS is still unclear, although many mechanisms of drug resistance have been well characterized. The present study was undertaken to investigate the effects of N-acetylcysteine (NAC), a GSH precursor, on the CDDP-induced effects in bladder cancer cells (KU1). The cytotoxic effects of CDDP were significantly blunted by NAC (1 mM) in KU1 cells. The IC50 of CDDP only (10.2+/-1.2 microM) is significantly lower than that of CDDP with NAC (IC50: 20.3+/-1.6 microM) in KU1 cells. NAC also significantly increased the intracellular concentration of GSH in KU1 cells (37.2+/-1.6 nmol/10(6) cells), compared to controls (15.9+/-7.6 nmol/10(6) cells). While CDDP produced a significant increase in ROS as measured in terms of dichlorofluorescein (DCF) production in KU1 cells in a time-dependent manner, pretreatment with NAC significantly reduced CDDP-induced intracellular DCF in KU1 cells. Moreover, TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay showed that CDDP-induced apoptosis (31.1+/-3.8%) was significantly inhibited by pretreatment with NAC in KU1 cells (11.2+/-2.6%). These results demonstrated that NAC scavenges CDDP-induced ROS and inhibits CDDP-induced cytotoxicity, suggesting that ROS mediate the CDDP-induced cytotoxicity in bladder cancer cells.     

Areca nut extract and arecoline induced the cell cycle arrest but not apoptosis of cultured oral KB epithelial cells: association of glutathione, reactive oxygen species and mitochondrial membrane potential

There are 600 million betel quid (BQ) chewers in the world. BQ chewing is a major etiologic factor of oral cancer. Areca nut (AN) and arecoline may inhibit the growth of oral mucosal fibroblasts (OMF) and keratinocytes. In this study, AN extract (100-800 microg/ml) and arecoline (20-120 microM) inhibited the growth of oral KB cells by 36-90 and 15-75%, respectively. Exposure to arecoline (> 0.2 mM) for 24 h induced G(2)/M cell cycle arrest of OMF and KB cells. Areca nut extract (> 400 microg/ml) also induced G(2)/M arrest of KB cells, being preceded by S-phase arrest at 7-h of exposure. No evident sub-G(0)/G(1) peak was noted. Marked retraction and intracellular vacuoles formation of OMF and KB cells were observed. Glutathione (GSH) level, mitochondrial membrane potential (Deltabetam) and H(2)O(2) production of KB cells were measured by flow cytometry. GSH level [indicated by 5-chloromethyl-fluorescein (CMF) fluorescence] was depleted by 24-h exposure of KB cells to arecoline (0.4-1.2 mM) and AN extract (800-1200 microg/ml), with increasing the percentage of cells in low CMF fluorescence. By contrast, arecoline (0.1-1.2 mM) and AN extract (800-1200 microg/ml) induced decreasing and increasing H(2)O(2) production (by 2',7'-dichloro- fluorescein fluorescence), respectively. Hyperpolarization of Deltabetam (increasing of rhodamine uptake) was noted by 24-h exposure of KB cells to arecoline (0.4-1.2 mM) and AN extract (800-1200 microg/ml). AN extract (100- 1200 microg/ml) and arecoline (0.1-1.2 mM) induced little DNA fragmentation on KB cells within 24 h. These results indicate that AN ingredients are crucial in the pathogenesis of oral submucous fibrosis (OSF) and oral cancer by differentially inducing the dysregulation of cell cycle control, Deltabetam, GSH level and intracellular H(2)O(2) production, these events being not coupled with cellular apoptosis.     

Docetaxel enhances the cytotoxicity of cisplatin to gastric cancer cells by modification of intracellular platinum metabolism

We have examined the combined anticancer effects of docetaxel (DOC) and cisplatin (CDDP) in vitro using the gastric cancer cell lines MKN-45, MKN-74, and TMK-1. Treatment of the cell lines with 30 microg/ml of DOC for 24 h followed by incubation with 3 or 10 microg/ml of CDDP for 24 h showed a clear synergistic effect. Sequence dependency of the agents was observed in these cell lines: DOC followed by CDDP (DC) showed a stronger antitumor effect than CDDP followed by DOC (CD) in all cell lines. To clarify the mechanism of action of the DC combination, total intracellular platinum (Pt) levels were evaluated after treatment with CDDP alone or combined with DC. For the MKN-45 and -74 cell lines, cells treated with DOC (10 microg/ml for 12 h) and then CDDP showed significantly increased intracellular Pt accumulation compared to cells treated with CDDP alone. We also investigated alterations in intracellular glutathione (GSH) concentration in response to DOC and CDDP. MKN-45 and -74 cells pretreated with DOC (10 microg/ml for 12 h) showed significantly increased intracellular GSH levels compared to cells administered CDDP only. To explain these findings, messenger RNA (mRNA) levels for multidrug resistance-associated protein-1 (MRP-1), the ATP-dependent pump for Pt-GSH complexes, were quantified in CDDP-treated MKN-45 cells with and without DOC pretreatment. While CDDP administration increased MRP-1 mRNA expression in MKN-45 cells, MRP-1 was not up-regulated after CDDP administration in DOC pretreated MKN-45 cells. Our results suggested that the enhanced CDDP toxicity due to DOC pretreatment may be related to the accumulation of intracellular Pt-GSH complexes, because DOC appears to suppress the MRP-1 up-regulation induced by CDDP exposure in gastric cancer cells.     

N-Acetylcysteine Modifies cis-Dichlorodiammineplatinum-induced Effects in Bladder Cancer Cells

We previously demonstrated a role of reactive oxygen species (ROS) in cytotoxicity induced by cis -dichlorodiammineplatinum (CDDP) in combination with glutathione (GSH) depletors in bladder cancer cells. However, the relationship between CDDP and ROS is still unclear, although many mechanisms of drug resistance have been well characterized. The present study was undertaken to investigate the effects of N-acetylcysteine (NAC), a GSH precursor, on the CDDP-induced effects in bladder cancer cells (KU1). The cytotoxic effects of CDDP were significantly blunted by NAC (1 m M ) in KU1 cells. The IC₅₀ of CDDP only (10.2±1.2 μM) is significantly lower than that of CDDP with NAC (IC₅₀: 20.3±1.6 μM) in KU1 cells. NAC also significantly increased the intracellular concentration of GSH in KU1 cells (37.2±1.6 nmol/10⁶ cells), compared to controls (15.9±7.6 nmol/10⁶ cells). While CDDP produced a significant increase in ROS as measured in terms of dichlorofluorescein (DCF) production in KU1 cells in a time-dependent manner, pretreatment with NAC significantly reduced CDDP-induced intracellular DCF in KU1 cells. Moreover, TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay showed that CDDP-induced apoptosis (31.1±3.8%) was significantly inhibited by pretreatment with NAC in KU1 cells (11.2±2.6%). These results demonstrated that NAC scavenges CDDP-induced ROS and inhibits CDDP-induced cytotoxicity, suggesting that ROS mediate the CDDP-induced cytotoxicity in bladder cancer cells.     

顺氨氯铂,阿霉素增强人抗CD3单克隆抗体活化杀伤细胞的杀瘤效应

应用抗CD3单克隆抗体和基因重组人IL-2共同诱导人外周血单个核细胞，制备抗CD3单克隆抗体活化的杀伤细胞（CD3AK）。利用LDH释放法，观察顺氨氯铂（CDDP）和阿霉素（ADM）预处理人肝癌细胞系H－7402，对CD3AK杀伤该靶细胞的调节作用。ABC－ELISA法检测CDDP和ADM对H-7402细胞表达细胞间粘附分子-1（ICAM-1）、HLA－ABC、HLA－DR抗原的影响。结果表明：经CDDP（2．0μg／ml）、ADM（O．1μg／ml）预处理12h的H－7402细胞对CD3AK的杀伤敏感性显著提高（P＜0.05）；CDDP能够明显促进H－7402细胞表达ICAM-1、HLA－ABC分子（P＜0.05）；ADM预处理的肿瘤细胞表面三种分子的表达均未发生明显的变化。CDDP促进人肝癌细胞对CD3AK的杀伤敏感性可能与其上调了肿瘤细胞表面的ICAM-1分子有关。     

Overcoming tnf-alpha and cddp resistance of a human ovarian-cancer cell-line (c30) by treatment with buthionine sulfoximine in combination with tnf-alpha and or cddp

Previous studies have demonstrated that glutathione (GSH) plays an important role in a wide range of cellular functions including protection, detoxification, transport and metabolism. GSH has been implicated in tumor cell resistance to drugs and/or cytotoxic factors. Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, depletes intracellular GSH and thus could reverse resistance. The present study investigated the effect of BSO used in combination with tumor necrosis factor-alpha (TNF-alpha) or cisdiamminedichloroplatinum (II) (CDDP) on cytotoxicity of a TNF-alpha and CDDP resistant human ovarian cancer cell line (C30). Cytotoxicity was monitored by the MTT assay. Treatment of C30 cells with BSO and CDDP or BSO and TNF-alpha resulted in overcoming resistance and a synergistic cytotoxic effect was obtained. Pretreatment of the tumor cells by either agent for 4 h and wash and followed by the addition of the second agent for 20 h resulted in the same cytotoxicity as observed in the presence of the two agents. Furthermore, combination treatment with BSO, CDDP and TNF-alpha further augmented the synergistic cytotoxic activity achieved by two agents against C30 cells. The protective effect of GSH was shown for TNF-alpha but not for CDDP as treatment of C30 cells with TNF-alpha in combination with GSH or N-acetyl-cysteine (NAC) reduced the cytotoxic effect of TNF-alpha. One mechanism of resistance to TNF-alpha in tumor cells is through the induction of TNF-alpha mRNA and/or protein. The C30 cells did not constitutively express TNF-alpha mRNA, however, treatment of C30 cells with TNF-alpha upregulated the expression of TNF-alpha mRNA. When BSO was used in combination with TNF-alpha, the level of TNF-alpha mRNA induced by TNF-alpha was markedly reduced. Further, incubation of C30 cells with TNF-alpha in conjunction with GSH or NAC also downregulated the expression of TNF-alpha mRNA induced by TNF-alpha. These findings demonstrate that treatment with BSO in combination with TNF-alpha or CDDP can overcome the resistance of C30 tumor cells to TNF-alpha and CDDP. The depletion of intracellular GSH and downregulation of TNF-alpha mRNA by BSO may play a role in the enhanced cytotoxicity seen with the combination of BSO and TNF-alpha. The synergistic effect obtained with a CDDP selected resistant ovarian cancer cell line suggests that treatment with BSO in conjunction with either TNF-alpha or CDDP, or TNF-alpha and CDDP may have a clinical application in the therapy of TNF-alpha and/or CDDP resistant ovarian tumors'     

Synergistic Enhancement of Cisplatin Cytotoxicity by SN-38, an Active Metabolite of CPT-11, for Cisplatin-resistant HeLa Cells

A cisplatin ( cis -diamininedichloroplatinuin(II); CDDP)-resistant HeLa cell line (HeLa/CDDP cells), which showed more than 8-fold resistance to CDDF compared to the parent cells, was newly established for this study. HeLa/CDDP cells accumulated 50% less platinum than the parent cells. There was no difference in intracellular glutathione (GSH) content between the parent and HeLa/ CDDP cells. The dose modification factor by DL-buthionine-S, R-sulfoximine (BSO) pretreatment was similar in both cell lines. HeLa/CDDP cells had cross-resistance to diammine (l, l-cyclobutanedicarboxylato)platinum(II) (CBDCA), ( cis -diammine (glycolato)platinum (254-S), but not to (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato)platinum(II) (DWA2114R), adriamycin, or VP-16. HeLa/CDDP cells showed a collateral sensitivity to 7-ethyl-10-hydroxycampto-thecin (SN-38), an active metabolite of 7-ethyl-10-[4-(l-piperidino)-l-piperidino]carbonyloxycamptothecin (CPT-11). Furthermore, isobologram analysis indicated synergistic interaction of CDDP and SN-38 only for HeLa/CDDP cells. The present study suggests that combination therapy with CDDP and CPT-11 may he potentially useful in the treatment of some patients with CDDP-resistant cancer.     

Glutathione restores normal cell activation and cell cycle progression in cis-platinum treated human lymphocytes

Cis-platinum (CDDP) induces severe inhibition of cell activation and cell cycle progression in PHA-stimulated human PBL's. Applying the novel BrdU/Hoechst flow cytometric technique for high resolution cell cycle analysis we show that CDDP induced multiple cell kinetic disturbances occur simultaneously comprising G0/G1-arrest, and slow down and arrest of cells in S and G2/M-phase. We investigated whether the administration of reduced glutathione (GSH) might rescue cells from proliferative disturbances induced by CDDP. GSH at 0.15 mg ml-1 only partially restored normal cell activation and cell cycle progression. However, at 1.5 mg ml-1 a complete normal proliferation pattern was obtained. At the highest GSH dose rescue from inhibition of cell activation (G0/G1-phase arrest) as well as of cell cycle progression (S- and G2/M-phase arrest) was also present after delayed addition of GSH (1, 4 and 20 h) to CDDP treated PBL's. In addition cell viability of CDDP exposed PBL's is restored after GSH treatment. Our in vitro experiments give evidence that an increase of WBC found in CDDP/GSH treated patients has a real underlying cellular physiological mechanism protecting human peripheral lymphocytes from CDDP toxicity.     

Modulation of cisplatin sensitivity and accumulation by interferon α-2A in human squamous carcinoma cell lines

This study was undertaken to elucidate the mechanism(s) of potentiation of cisplatin (CDDP) cytotoxicity by interferon α-2a (IFN α-2a) in human squamous carcinoma cell lines SCC-25 and SCC-4. IFN α-2a treatment significantly increased the cytotoxicity of CDDP in both cell lines in a dose-dependent manner. In SCC-25 cells, the cytotoxicity of CDDP was increased by about 2- and 4-fold, respectively, by treating the cells with 400 and 800 1U/ml IFN α-2a. Sensitivity of SCC-4 cells to CDDP was increased by about 3- and 7-fold, respectively, by 400 and 800 1U/ml IFN α-2a treatment. Drug uptake experiments revealed approximately 1.4- to 5-fold higher platinum accumulation in IFN α-2a-treated cells as compared to respective controls. Cellular levels of glutathione (GSH) and GSH transferase, which have been suggested to be important determinants of tumor cell sensitivity to CDDP, were not altered by IFN α-2a treatment in either of the cell lines. Northern blot analysis showed a moderate increase (about 30–40%) in the level of MT-II_A mRNA by IFN α-2a treatment in these cells. Our results suggest that IFN α-2a-mediated sensitization of SCC-25 and SCC-4 cell lines to CDDP in vitro may be due to an increase in intracellular platinum accumulation.     

Fundamental study of combination chemotherapy with THP, 5-FU and CDDP for human KB carcinoma cell line and its multidrug resistant cell line KB-C1--usefulness of treatment with 5-FU preceding CDDP

This study was designed to investigate the usefulness of treatment with 5-FU preceding CDDP in combination chemotherapy with THP, 5-FU and CDDP. Using the human KB carcinoma cell line and its multidrug resistant cell line KB-C1, the difference in the antitumor effect due to the sequence of administration of CDDP and 5-FU (TCF or TFC) was examined on cultured cells and nude mouse tumor xenografts. When KB and KB-C1 were treated with THP (0.01 microgram/ml) on day 1, CDDP (0.05 microgram/ml) on day 2 or day 4 and 5-FU (0.25 microgram/ml) on day 3 and 4 or day 2 and 3, TFC suppressed the cell proliferation more strongly than TCF (p < 0.05), though there was no difference between KB and KB-C1. In nude mouse xenografts, intraperitoneal administrations of THP (0.5 mg/kg) on day 1, CDDP (2 mg/kg) on day 2 or day 5, and 5-FU (10 mg/kg) on day 3-5 or day 2-4 inhibited tumor growth more effectively in KB than in KB-C1. At three weeks postadministration, growth inhibition by TCF and TFC was 29.9% and 57.4% in KB and 25.2% and 49.8% in KB-C1, respectively. These results indicate that TFC was superior to TCF in cytocidal and antitumor effects for KB and KB-C1.     

Chemosensitization of oral squamous cell carcinoma cells to cisplatin by histone deacetylase inhibitor, suberoylanilide hydroxamic acid

In the present study, the precise mechanism of the enhancing action of histone deacetylase (HDAC) inhibitors on cisplatin (CDDP)-induced apoptosis was investigated using suberoylanilide hydroxamic acid (SAHA) in human oral squamous cell carcinoma cells (HSC-3). HDAC inhibitors are promising novel compounds for the treatment of cancer, which cooperate with chemotherapeutic agents to induce apoptosis. Apoptosis enhancement of HSC-3 cells by SAHA was accompanied by the activation of caspase-3, -8 and -9, suggesting a mitochondrial-dependent amplification loop. Concomitant treatment (CDDP/SAHA) of cells resulted in the most effective enhancement of apoptosis compared to other timing combinations. By means of cell-cycle synchronization, G0/G1-phase cells proved to be a more sensitive fraction to SAHA action than their synchronized counterparts in other phases. Furthermore, cells treated with SAHA revealed a decrease in intracellular reduced glutathione (GSH) contents. Of importance, the GSH synthesis inhibitor, diethyl maleate, decreased intracellular GSH and enhanced CDDP-induced apoptosis in a similar pattern of timing to SAHA. Thus, SAHA appears to disrupt the intracellular redox balance, which causes maximal apoptosis at the G0/G1 phase arrested by CDDP in HSC-3 cells. These results demonstrate that HDAC inhibitors not only cause a change in the histone acetylation status, but are also able to influence the apoptotic process at several levels, and GSH plays a key role in governing SAHA-dependent enhancement of CDDP-induced apoptosis.     

Schedule-dependent reversion of acquired cisplatin resistance by 5-fluorouracil in a newly established cisplatin-resistant HST-1 human squamous carcinoma cell line

In vitro continuous stepwise exposure of HST-I human squamous carcinoma cell line to cisplatin (CDDP) for 12 months resulted in a 3.5-fold stably resistant subline designated HST-I/CPO.2. Compared with parental cells, this cell line showed a 1.8-fold increase in cellular glutathione (GSH) and a 50% reduction in initial numbers of DNA interstrand cross-links (ICLs), despite similar levels of intracellular platinum accumulation. Evaluation of the kinetics of DNA ICL removal at nearly equivalent levels of DNA ICL formation indicated that HST-I/CPO.2 cells appeared to remove DNA ICLs more rapidly than do HST-I parental cells. Thus, both elevated cellular GSH and increased DNA repair capacity would be the major factors contributing to CDDP resistance. Pretreatment of HST-I/CPO.2 cells with 5-FU, with drug-free intervals of 24 to 48 hr before exposure to CDDP, completely reversed CDDP resistance, or even increased the sensitivity to a level greater than that of parental cells, whereas the opposite sequence had no effect on resistance. In parallel with augmentation of the cytotoxicity, the levels of cellular GSH were significantly reduced over 48 hr by 5-FU pretreatment. However, depletion of cellular GSH using buthionine sulfoximine resulted in partial reversal of CDDP resistance, indicating that reduction of cellular GSH alone is not sufficient for complete reversal of CDDP resistance. Our data, together with evidence that 5-FU modulates the repair of platinum-DNA cross-links, suggest that schedule-dependent, complete reversal of CDDP resistance by 5-FU might be attributed to its inhibitory effects on both GSH levels and the repair of platinum-DNA adducts. Thus, optimization for the drug administration schedule is important when aiming at therapeutic synergy and circumvention of acquired CDDP resistance. © 1996 Wiley-Liss, Inc.     

In vitro susceptibility to anticancer agents of the human KB carcinoma cell line transfected with COX-2 cDNA

In order to investigate the malignant phenotype of cyclooxygenase (COX)-2 overexpressing cancer cells, a human epidermoid KB carcinoma cell line minimally expressing COX-2 protein was transfected with human COX-2 cDNA. In this study, we used a COX-2 transfected clone KB/COX-2 and a neomycin-transfected clone KB/neo as the control. When we examined the susceptibility to anticancer agents, there was no difference between these two clones in vincristine, bleomycin and 5-fluorouracil, although KB/COX-2 showed a 2.5-fold resistance to cisplatin (CDDP) as compared with KB/neo. The IC50 for CDDP was 4.3 microM in KB/COX-2 and 1.7 microM in KB/neo. Treatment with small interfering RNA (siRNA) mediated the inhibition of COX-2 significantly increasing the level of susceptibility to CDDP in COX-2 siRNA as compared to that of the control siRNA. The expression of MRP1 and MRP2 was stronger in KB/COX-2 than in KB/neo by Western blot analysis. In addition, apoptosis induction by CDDP was at a lower level in KB/COX-2 (31%) than in KB/neo (38%). These results suggested that the overexpression of COX-2 increases the intracellular production of MRP1 and MRP2 and causes drug resistance to CDDP.     

Enhancement of glucuronosyl etoposide transport by glutathione in multidrug resistance-associated protein-overexpressing cells

Multidrug resistance-associated protein (MRP) has been shown to transport glutathione (GSH) S-conjugates such as leukotriene C4 (LTC4) and S-(2,4-dinitrophenyl)-glutathione (DNP-SG). On the other hand, it has while it has been reported that MRP-overexpressing cells exhibit decreased sensitivity to drugs which do not form GSH S-conjugates. In this study, we found that GSH affects the transport of glucuronosyl etoposide as a major metabolite of etoposide in MRP-overexpressing KB/VP-4 cells. The relative resistance level of KB/VP-4 cells to etoposide was 70-fold that of wild-type KB cells. Membrane vesicles prepared from KB/VP-4 cells exhibited markedly enhanced ATP-dependent transport of glucuronosyl etoposide as well as LTC4. Transport of glucuronosyl etoposide was augmented in the presence of GSH. Treatment of KB/VP-4 cells with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, resulted in about 75% depletion of cellular GSH levels, a four-fold increase of the sensitivity to etoposide and depression of glucuronosyl etoposide efflux. These results suggest that GSH plays a role in the enhancement of MRP-mediated glucuronosyl etoposide transport.     

Using Acetaminophen's Toxicity Mechanism to Enhance Cisplatin Efficacy in Hepatocarcinoma and Hepatoblastoma Cell Lines

Background/Aims

Acetaminophen overdose causes hepatotoxicity mediated by toxic metabolites generated through the cytochrome P450 enzyme. The objective of this study was to investigate whether acetaminophen (AAP) can enhance cisplatin (CDDP) cytotoxicity against human hepatocarcinoma and hepatoblastoma cells in vitro and whether this effect can be prevented by N-acetylcysteine (NAC).

Methods

In vitro studies (glutathione [GSH] level, cell viability, and immunoblot assays) were performed using human hepatocarcinoma and hepatoblastoma cells cultured in AAP, CDDP, and the combination of both with or without delayed NAC administration. The pharmacology and toxicology of high-dose AAP in rats were also examined.

Results

Acetaminophen decreased GSH levels in liver cancer cells in a dose- and time-dependent manner. Acetaminophen combined with CDDP had enhanced cytotoxicity over CDDP alone. The cytotoxicity caused by AAP plus CDDP was decreased by NAC, with the effectiveness being time-dependent. The GSH level was lowered in the liver but not in the blood or the brain in rats treated with a high dose of AAP (1000 mg/kg). The expression of CYP2E1 protein, a key cytochrome P450 enzyme, varies among species but is not correlated to AAP sensitivity in liver cancer cells.

Conclusions

Our results suggest that a chemotherapeutic regimen containing both AAP and CDDP with delayed NAC rescue has the potential to enhance chemotherapeutic efficacy while decreasing adverse effects. This would be a promising approach particularly for hepatoblastomas regardless of cellular CYP2E1 protein level but could also be beneficial in other malignancies.     

Modulation of sensitivity to cis-diamminedichloroplatinum (II) by thromboxane A2 receptor antagonists in non-small-cell lung cancer cell lines.

We examined the effect of selective thromboxane A2 (TXA2) receptor antagonists, calcium 5(Z)-1R, 2S, 3S, 4S-7-[3-phenylsulphonylaminobicyclo [2.2.1] hept-2-yl]-5-heptonoate hydrate (S-1452) and +/- -7-(3,5,6,-trimethyl-1,4-benzoquinon-2-yl)-7-phenylhaptanoic acid (AA-2414), on sensitivity to cis-diamminedichloroplatinum (II) (CDDP) in non-small-cell lung cancer cell lines. IC50 values to CDDP using MTT assay were decreased 2.1- and 4.6-fold respectively by treatment with 250 or 500 microM S-1452, for a 2 h simultaneous drug exposure, and those of PC-9/CDDP, a CDDP-resistant cell line, were decreased 3.1- and 6.1-fold. Sensitivity to carboplatin was also enhanced by the treatment with S-1452. IC50 values to CDDP and carboplatin were decreased by treatment with AA-2414 in a dose-dependent manner. Isobologram analysis showed that the combination of CDDP with S-1452 or AA-2414 produced supra-additive or additive effects in each cell line. Neither glutathione content nor glutathione S-transferase activity was changed in either cell line by treatment with 500 microM S-1452. Accumulation of platinum into PC-9 and PC-9/CDDP was increased by the treatment in a dose-dependent manner. Na+, K+-ATPase activity of PC-9 and PC-9/CDDP was enhanced by the treatment of S-1452 in a dose-dependent manner. These data show that the TXA2 receptor antagonists may enhance the sensitivity of non-small-cell lung cancer cell lines to platinum agents. Increase in Na+, K+-ATPase activity induced by S-1452 may be the mechanism of its sensitising effect through increase in platinum accumulation.     

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.