CDDP-resistant Cell Line Research Articles

Expression of glutathione S-transferase-pi and sensitivity of human gastric cancer cells to cisplatin.

The authors examined the correlation between the expression of glutathione S-transferase-pi (GST pi) and the sensitivity of gastric cancer to anticancer agents. In 62 human gastric carcinomas, the expression of GST pi was immunohistochemically evaluated, and sensitivity to the anticancer drugs, cisplatin (CDDP), doxorubicin (DXR) aclacinomycin A (aclarubicin), (ACR), 5-fluorouracil (5-FU), mitomycin C (MMC) and carboquone (carbazilquinon) (CQ) was examined using the in vitro succinate dehydrogenase inhibition test. The authors used the Chinese hamster ovary (CHO) cell line and its variant CDDP-resistant cell line C/CDP-2, and they analyzed the relationship among CDDP-sensitivity, mRNA expression, and immunohistochemical staining. Immunohistochemically detected GST-pi-positive tumors were noted in 35 of 62 excised tumors. There was no significant correlation between GST-pi expression and clinicopathologic features or prognosis. The succinate dehydrogenase activity of each drug for tumors, with regard to negative or positive GST-pi, was 34.9 plus or minus 13.7% and 46.0 plus or minus 22.0% for CDDP, with a significant statistical difference (P < 0.05). However, there was no statistical difference for the other drugs tested. C/CDP-2 cells showed a lower sensitivity to CDDP, a higher expression of mRNA for GST pi and a stronger immunohistochemical staining than CHO cells. The overexpression of GST-pi is significantly related to the sensitivity of gastric cancer to CDDP.

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'

Differential macrophage-mediated cytotoxicity to P388 leukemia cells and its drug-resistant cells examined by a new MTT assay

After activation by interferon-γ (INF-γ) and lipopolysaccharide(LPS), mouse peritoneal macrophages were cocultured with P388 parental cell line(P388/PRT) and its adriamycin(ADM)-, cisplatin(CDDP)-, cyclophosphamide(CPM)-, and mitomycin-C(MMC)-resistant cell lines for one day at effector:target ratios(E:T) of 10:1, 5:1, and 2:1. The direct 3-(4,5-dimethylthiazol-2-yl)2,5diphenyl tetrazolium bromide (MTT) cleavage assay and a new indirect MTT assay as well as clonogenic assay were used to quantitate activated macrophage-mediated cytotoxicity to these nonadherent leukemia targets. The results revealed that all the P388 cell lines can be suppressed efficiently by activated macrophages, but P388 CPM- and MMC-resistant cell lines (P388/CPM, P388/MMC) were more susceptible than P388/PRT while P388 ADM- and CDDP-resistant cell lines (P388/ADM, P388/CDDP) shared equal level of survival rates with P388/PRT. This study also showed that both non-activated and activated macrophages can produce formazan in a high level, which can interfere with the final results of direct MTT assay. The new indirect MTT assay can avoid such interference by separating the effectors from the targets before performing the MTT assay and reflects the real viability of the targets so the indirect MTT assay developed in this study could be a better way to examine cytostatic and cytotoxic effect of activated macrophages an non-adherent tumor cells in vitro.

Increased phosphorylation of nuclear phosphoproteins in human lung-cancer cells resistant to cis-diamminedichloroplatinum (II).

A novel non-phorbol-ester-like tumor promoter, okadaic acid (OA) has been shown to be an inhibitor of protein phosphatase I and IIA and, thus, to cause an "apparent activation" of protein kinase C (PKC). We previously showed that cis-diamminedichloroplatinum(II) (CDDP)-resistant cells, PC-9/CDDP, were cross-resistant to OA and that the cross-resistance was not due to the increased efflux of OA. We hypothesized that the phosphorylation status of some cellular proteins might be important in CDDP-resistance. No significant difference in PKC activity or total protein phosphatase activity measured in vitro was seen between PC-9 and PC-9/CDDP cells, nor in their sensitivity to inhibition by OA, nor in the amount of phosphorylation of whole cells or TCA-insoluble material. By SDS-PAGE after incubation of intact cells with 32P, we detected a marked increase, compared to PC-9 cells, in phosphorylation of the nuclear proteins of MW 32 and 20 kDa in CDDP-resistant PC-9/CDDP cells with no apparent difference in protein content. When phosphorylation of nuclear proteins observed in PC-9/CDDP cells was analyzed by 2-dimensional SDS-PAGE, the 32-kDa protein had a PI of about 4.5. The 32-kDa and 20-kDa bands were increased in a dose-dependent manner by CDDP treatment. On the other hand, no increase in phosphorylation of these proteins was observed in parental PC-9 cells. These results demonstrate a marked difference in the phosphorylation status of specific nuclear proteins between parental and CDDP-resistant cell lines, which may be related to CDDP-resistance.

Accumulation of cis-diamminedichloroplatinum (II) and its analogues in sensitive and resistant human ovarian carcinoma cells.

Human ovarian carcinoma cell line (NOS2), established from a patient with serous cystadenocarcinoma of the ovary, has been exposed to a stepwise increase in cis-diamminedichloroplatinum (II) (CDDP) concentration to produce a CDDP-resistant cell line NOS2CR) as an experimental model for resistance to CDDP. NOS2CR cells showed a 7-fold resistance to CDDP and a lesser degree of cross-resistance to diammine (1,1-cyclobutanedicarboxylato)-platinum (II) (CBDCA) and (-)-(R)-2-aminomethylpyrrolidine (1,1-cyclobutanedicarboxylato) platinum (II) (DWA2114R). In the absence of CDDP, cross-resistance to DWA2114R was reduced to the original level by 2 months, although 83% resistance to CDDP remained up to 6 months. To investigate CDDP-resistant mechanisms, alterations in the intracellular accumulation of CDDP and analogues were assayed by atomic absorption. In both NOS2 and NOS2CR cells, accumulation of CDDP increased linearly with time and was concentration-dependent. NOS2CR cells demonstrated 71, 52 and 12% reduction in accumulation of CDDP, CBDCA, and DWA2114R, respectively. These reductions did not seem to be due to P-glycoprotein, because expression of multidrug-resistant 1 gene was not detected in either NOS2 or NOS2CR cells. These studies indicate that the mechanisms of resistance to CDDP and analogues in NOS2CR cells are related in the main to reduced intracellular accumulation of drugs. DWA2114R might be helpful to treat CDDP-resistant and recurrent tumors which were treated by CDDP.

Combination of the minor groove-binder U73-975 or the intercalator mitoxantrone with antitumor alkylating agents in MCF-7 or MCF-7/CP cells

In an effort to improve the cytotoxicity of clinically used anticancer alkylating agents, the topoisomerase II inhibitory drugs U73-975 or mitoxantrone were added to cell cultures exposed to CDDP, carboplatin, BCNU, melphalan or thiotepa. In the MCF-7 human breast cancer cell line and in the MCF-7/CP (CDDP resistant) subline, U73-975 and mitoxantrone were both potent cytotoxic agents (IC 50 0.002 μM and 0.006 μM for U73-975, respectively and 0.8 μM and 0.1 μM for mitoxantrone, respectively). As evaluated by isobologram analysis, the addition of either U73-975 or mitoxantrone to 1 h exposure to CDDP resulted in greater-than-additive killing in the MCF-7 parent cells. While U73-975 was also greater-than-additive in cytotoxicity with CDDP in the MCF-7/CP line, mitoxantrone and CDDP were only additive in cytotoxicity in these cells. In the case of carboplatin, the addition of U73-975 or mitoxantrone to treatment with the drug resulted in greater-than-additive cell killing in the MCF-7 parental cell line but in the MCF-7/CP cell line these combinations were only additive in cell killing. Addition of U73-975 to treatment with BCNU resulted in only additive cytotoxicity in both cell lines; however, the combination of mitoxantrone with BCNU resulted in greater-than-additive cell killing in both the parental and CDDP resistant cell lines. When either U73-975 or mitoxantrone was added to treatment with melphalan greater-than-additive cytotoxicity resulted in both cell lines except at low melphalan concentrations in the MCF-7/CP cell line. Finally, the addition of either modulator to treatment with thiotepa in the MCF-7 cell line produced variable interactions depending on thiotepa concentration, but in the MCF-7/CP cell line either modulator in combination with thiotepa caused greater-than-additive cell killing. These results indicate that the addition of topoisomerase II inhibitory drugs may substantially increase the cytotoxicity of some alkylating agents. In vivo experiments are necessary, however, to ascertain whether a therapeutic gain is achievable.

The Relationship of the Proliferating Cell Nuclear Antigen Protein to &lt;i&gt;cis&lt;/i&gt;-Diamminedichloroplatinum(II) Resistance of a Murine Leukemia Cell Line P388/CDDP

We investigated whether the proliferating cell nuclear antigen (PCNA) protein takes part in cis-diamminedichloroplatinum (II) (CDDP) resistance, using a murine leukemia cell line P388 and its CDDP resistant cell line. P388/CDDP was 4 times more resistant to CDDP than P388. The cell lines were maintained in the DBA/2 female mouse peritoneal space. In total cells, the amount of the PCNA protein decreased to 90% in P388 after 1 h CDDP treatment, but that of P388/CDDP increased to 127%. The difference was statistically significant (p = 0.012, n = 5). As for G2/M phase cells, the difference was also significant at 1 h (p = 0.016, n = 5) and at 2 h (p = 0.036, n = 5) after treatment. In P388 the amount of the PCNA protein decreased in accordance with the inhibited cell proliferation, whereas in P388/CDDP, the amount of the PCNA protein increased in spite of the inhibited cell proliferation. This increase of the PCNA protein suggests that the PCNA protein is involved in CDDP resistance of P388/CDDP through enhanced DNA repair synthesis.

Augmentation of CDDP cytotoxicity by amphotericin B in CDDP resistant human lung cancer cell lines in vitro and in vivo: Morikage, T., Bungo, M., Ohmori, T., Nishio, K., Fujiwara, Y. and Saijo, N. Pharmacology Division, National Cancer Center Research Institute, Tsukiji, Chuoku, Tokyo 104, Japan

Augmentation of CDDP cytotoxicity by amphotericin B in CDDP resistant human lung cancer cell lines in vitro and in vivo: Morikage, T., Bungo, M., Ohmori, T., Nishio, K., Fujiwara, Y. and Saijo, N. Pharmacology Division, National Cancer Center Research Institute, Tsukiji, Chuoku, Tokyo 104, Japan

Possible Mechanisms of Resistance to cis‐Diamminedichloroplatinum (II) of Human Ovarian Cancer Cells

The mechanism of cis-diamminedichloroplatinum (II) (CDDP) resistance was examined by using a CDDP-resistant (KFr) cell line established by continuous exposure of KF cells (derived from serous cystadenocarcinoma of the ovary) to escalating doses of CDDP. When KFr cells were incubated with 66.7 microM CDDP, the uptake of CDDP was significantly inhibited and the cellular content in the KFr cells was about a half of that in KF cells after incubation for 4 h. When the KF or KFr cells were incubated for 4 h with 100 microM CDDP, the release pattern of CDDP from KFr cells was similar to that from KF cells. In addition, the DNA histogram of both KF and KFr cells revealed that KF cells seemed to contain two clones of cell population and the KFr cells may have been selected by exposure to CDDP. At 3 h after intraperitoneal administration of 0.5 mg of CDDP per mouse to nude mice with KF or KFr tumor, the CDDP content in the KFr tumor was significantly lower than that in the KF tumor. In contrast, at 6 or 9 h after CDDP administration the CDDP content in the KFr tumor was significantly higher than that in the KF tumor. Furthermore, the KFr cells had cross-resistance to various CDDP analogues including carboplatin. It was shown that cellular uptakes of two CDDP analogues into KFr cells were significantly lower than those into KF cells.

The use of oxaliplatin versus cisplatin in intraperitoneal chemotherapy in cancers restricted to the peritoneal cavity in the rat

Pharmacokinetic studies demonstrated the advantage of intraperitoneal oxaliplatin (1-OHP) for cancers restricted to the peritoneal cavity. The area under the concentration X time curve (AUC) in the peritoneal cavity for both total and ultrafiltered drug was almost 2 times higher for 1-OHP than cisplatin (cDDP). The AUC for ultrafiltered 1-OHP in plasma was also a factor 4 higher than cDDP, indicating that peritoneal tumors received a higher exposure from 1-OHP than cDDP directly in the peritoneal cavity and indirectly via the systemic circulation. Total platinum concentrations in peritoneal tumors of rats were determined after i.p. administration of equimolar doses of 1-OHP and cDDP. In spite of the pharmacological advantages, no significant difference in platinum concentration was demonstrated. In addition, no difference in the distribution of platinum within peritoneal tumors was detected after i.p. treatment with equimolar doses, i.e., platinum concentrations were comparable both in the periphery, 29 +/- 4 ppm for cDDP and 22 +/- 8 for 1-OHP and in the center of the tumor, 18 +/- 3 for both drugs. When CC531 tumor cells were incubated in vitro with equimolar concentrations of 1-DHP and cDDP in vitro, 2 to 4 times less platinum was found in cells treated with 1-OHP, indicating that the uptake of 1-OHP differed from that of cDDP. Oxaliplatin was not cross resistant for cDDP in CC531.RL4 tumor cells, a cDDP resistant cell line, which may indicate its value in ovarian cancer patients who did not respond to earlier cDDP treatment.

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.