Randolph D. Christen

Synergistic interaction between cisplatin and taxol in human ovarian carcinoma cells in vitro.

Taxol, a unique tubulin active agent, was found to demonstrate a marked schedule-dependent synergistic interaction with cisplatin (DDP) in the killing of human ovarian carcinoma 2008 cells in vitro as determined by median effect analysis. The interaction was highly synergistic when 19 h taxol exposure was followed by 1 h concurrent exposure to taxol and DDP. The combination indices (CIs) on this schedule were 0.11 +/- 0.1, 0.25 +/- 0.15 and 0.39 +/- 0.14 at 20%, 50% and 80% cell kill respectively. However, the interaction was antagonistic when 1 h exposure to DDP was followed by 20 h exposure to taxol, or when cells were exposed to DDP and taxol for 1 h concurrently. When taxol preceded DDP, synergy was also observed with the 11-fold DDP-resistant 2008/C13*5.25 subline, which yielded CI values of 0.21 +/- 0.02, 0.30 +/- 0.11 and 0.31 +/- 0.17 at 20%, 50% and 80% cell kill respectively. At an IC50 concentration, taxol had no effect on [3H]cis-dichloro(ethylenediamine) platinum uptake, on the permeability of the plasma membrane or on glutathione or metallothionein levels in 2008 or 2008/C13*5.25 cells. Mitotic arrest in these cells was observed only at taxol concentrations well above those required for synergy with DDP, suggesting that the mechanism underlying the synergistic interaction was not a taxol-induced alteration in cell cycle kinetics. Of additional interest was the fact that the 2008/C13*5.25 cells were hypersensitive to taxol, and that this was partially explained by an alteration in the biochemical pharmacology of taxol. Although cellular taxol accumulation reached steady state within 2 h in both cell lines, taxol efflux was slower and the taxol was more extensively bound in 2008/C13*5.25 cells than in 2008 cells. In addition, the 2008/C13*5.25 cells had only 55% of the parental levels of beta-tubulin content. However, in another pair of DDP-sensitive and -resistant ovarian cell lines no taxol hypersensitivity and no change in beta-tubulin content was observed, indicating that the DDP-resistant and taxol-hypersensitive phenotypes do not segregate together. We conclude that taxol interacts synergistically with DDP in a manner that is highly schedule dependent, and that the hypersensitivity of 2008/C13*5.25 cells no taxol is unrelated to the mechanism of synergy. These in vitro observations suggest that drug schedule will be an important determinant of the activity and toxicity of the DDP and taxol drug combination in clinical studies.

Induction of JNK and c-Abl signalling by cisplatin and oxaliplatin in mismatch repair-proficient and -deficient cells.

SummaryLoss of DNA mismatch repair has been observed in a variety of human cancers. Recent studies have shown that loss of DNA mismatch repair results in resistance to cisplatin but not oxaliplatin, suggesting that the mismatch repair proteins serve as a detector for cisplatin but not oxaliplatin adducts. To identify the signal transduction pathways with which the detector communicates, we investigated the effect of loss of DNA mismatch repair on activation of known damage-responsive pathways, and recently reported that cisplatin differentially activates c-Jun NH2-terminal kinase (JNK) and c-Abl in repair-proficient vs.-deficient cells. In the current study, we directly compared differential activation of these pathways by cisplatin vs. oxaliplatin. The results confirm that cisplatin activates JNK kinase 5.7 ± 1.5 (s.d.)-fold more efficiently in DNA mismatch repair-proficient than repair-deficient cells, and that the c-Abl response to cisplatin is completely absent in DNA mismatch repair-deficient cells. In contrast, there was no detectable activation of the JNK or c-Abl kinases in DNA mismatch repair-proficient or -deficient cells exposed to oxaliplatin. The present study demonstrates that, despite the similarity of the adducts produced by cisplatin and oxaliplatin, they appear to be recognized by different detectors. The DNA mismatch repair system plays an important part in the recognition of cisplatin adducts, and activation of both the JNK and c-Abl kinases in response to cisplatin damage is dependent on the detector function of the DNA mismatch repair proteins. In contrast, this detector does not respond to oxaliplatin adducts.

Enhancement of drug sensitivity of human malignancies by epidermal growth factor

We have previously shown that epidermal growth factor (EGF) enhances the in vitro and in vivo sensitivity of human ovarian carcinoma 2008 cells to cisplatin. EGF was found to enhance selectively the in vivo toxicity of cisplatin to 2008 cell xenografts without altering the toxicity of cisplatin to non-malignant target tissues such as the kidney or bone marrow. We now show that recombinant human EGF (rhEGF) enhances the cisplatin sensitivity of cell lines representative of many other types of malignancies in addition to ovarian carcinoma, including cancers of the head and neck, cervix, colon, pancreas and prostate, as well as non-small-cell carcinoma of the lung. In addition, rhEGF was found to sensitise cells to other platinum-containing drugs and several other classes of chemotherapeutic agents. rhEGF sensitised 2008 cells not only to cisplatin, but also to carboplatin and tetraplatin, as well as taxol, melphalan and 5-fluorouracil. We conclude that modulation of drug sensitivity by rhEGF is observed in cell lines representative of many human malignancies and for multiple classes of chemotherapeutic agents, indicating that it alters one or more components of the cellular damage response that are both common between cell lines and classes of drugs and fundamental to survival.

Tamoxifen delays the development of resistance to cisplatin in human melanoma and ovarian cancer cell lines

The development of resistance to cisplatin (DDP) occurs rapidly both in vitro and in vivo, and constitutes a major obstacle to effective therapy. We have previously demonstrated that there is a highly synergistic interaction between tamoxifen (TAM) and DDP against cell lines representative of three different human cancers: melanoma, ovarian carcinoma and small-cell lung cancer. The purpose of these studies was to determine if TAM interferes with the development of resistance to DDP. T-289 melanoma cells and 2008 ovarian cancer cells were cultured with increasing concentrations of DDP +/- TAM in an attempt to induce resistance to DDP. At various time points the cells were removed from culture and the degree of resistance to DDP was quantitated. Concurrent exposure to TAM and DDP decreased both the rate and the absolute magnitude of resistance to DDP in both melanoma and ovarian cancer cell lines. In the T-289 cell line the rate was decreased by a factor of 3.4 +/- 1.4 (P < 0.05), while in the 2008 cell line the rate was decreased by a factor of 2.4 (P < 0.01). TAM decreases the rate as well as the absolute magnitude of in vitro resistance to DDP in both melanoma and ovarian cancer cell lines. These data suggest that the concurrent administration of TAM and DDP may result in a delay in the development of resistance to DDP which may have important clinical implications in the design of DDP-containing regimens.

Induction of the growth arrest and DNA damage-inducible gene GADD153 by cisplatin in vitro and in vivo.

The inability to assess the extent of tumour damage immediately following treatment is a major clinical obstacle to improving the management of cancer patients. Normally, the effectiveness of chemotherapy or radiation therapy cannot be determined for at least several weeks after treatment. We studied the increase in mRNA of the growth arrest and DNA damage-inducible gene GADD153 in human 2008 ovarian carcinoma cells in vitro and in vivo to determine whether treatment-induced increases in the level of GADD153 mRNA could be used as a marker of the extent of tumour damage. GADD153 mRNA was increased in a transient, dose-dependent manner by cisplatin (DDP) when the tumour cells were grown both in vitro and as tumour xenografts in nude mice. The magnitude of induction of GADD153 mRNA did not vary significantly between different 2008 xenografts treated with equal doses of DDP, and GADD153 mRNA induction correlated with the degree of in vitro cytotoxicity for two different schedules of drug exposure. DDP increased GADD153 mRNA levels in melanoma and head and neck xenograft models as well. We conclude that the increase in GADD153 mRNA can be used to detect tumour injury at time points as short as 24 h after administration of DDP.

Phase I and pharmacokinetic study of intraperitoneal topotecan

Objective. To determine the maximum tolerated dose and pharmacokinetics of topotecan when administered by the intraperitoneal route. Methods. A dose-escalating Phase I trial was conducted in which fifteen % of the total dose was given as an intraperitoneal bolus in two litres of D5W and the remainder was given as a continuous intraperitoneal infusion over 24 hours. Treatments were given every 21 days. Pharmacokinetic analyses were performed at the recommended phase II dose. Results. Seventeen patients received a total of 43 cycles at 21-day intervals. The maximum tolerated dose was 4 mg/m2 and acute dose-limiting toxicity was neutropenia. Other toxicities included leukopenia, anemia, emesis, fever, and abdominal pain. Although no objective responses were achieved, five of ten patients with ascites had a decrease in fluid accumulation with administration of intraperitoneal topotecan. The recommended phase II dose is 3 mg/m2. Pharmacokinetic analysis performed at a dose of 3 mg/m2 demonstrated that elimination from the peritoneal cavity followed second-order kinetics with k1 = 1.6 hr-1, k2 = 0.3 hr-1 and first and second-phase half-lives of 0.49 and 2.7 hours, respectively. Plasma pharmacokinetic behavior was best described by first-order kinetics with k = 0.5 hr-1 and a half-life of 3.9 hours. The pharmacologic advantage, expressed as the peritoneal to plasma AUC ratio was 31.2. Conclusions. Intraperitoneal administration of topotecan at 3 mg/m2 results in a substantial increase in drug exposure for the peritoneal cavity without compromising systemic exposure; this may be beneficial for the treatment of patients with ovarian cancer or intraperitoneal carcinomatosis.

Signaling and drug sensitivity.

Even though alterations in receptor and nonreceptor kinases are involved in the development of human cancer, many cancer cell lines still retain their responsiveness to growth factors. We have investigated the hypothesis that cellular signaling events regulate the sensitivity of cancer cells to chemotherapeutic agents. In 2008 human ovarian carcinoma cells, activation of a number of different transduction pathways resulted in a 2 to 4-fold increase in the sensitivity to cisplatin. These signaling events include pathways activated by the epidermal growth factor (EGF) receptor, tumor necrosis factor α (TNFα) receptor, bombesin receptor, protein kinase A (PKA), and protein kinase C (PKC). Enhanced sensitivity to chemotherapeutic agents is presumed to be mediated by phosphorylation of critical target protein(s). β-tubulin has been identified as one such target for the protein kinase A signaling cascade. For other signal transduction pathways the key substrates that regulate drug sensitivity have not yet been identified. Recent work has shown that DNA damaging agents activate signaling cascades one of which involves the Src, Ras, and Raf proteins as intermediates and results in induction of a number of genes, including c-fos, c-jun, and the growth arrest and DNA damage??inducible (gadd) genes. This signaling cascade has been shown to involve activation of protein kinase C and to have a protective function. With the growing understanding of how signaling events relate to damage response and drug sensitivity, new and potentially useful strategies for modulating drug sensitivity are evolving.

Cisplatin and taxol activate different signal pathways regulating cellular injury-induced expression of GADD153.

Signal transduction pathways activated by injury play a central role in coordinating the cellular responses that determine whether a cell survives or dies. GADD153 expression increases markedly in response to some types of cellular injury and the product of this gene causes cell cycle arrest. Using induction of GADD153 as a model, we have investigated the activation of the cellular injury response after treatment with taxol and cisplatin (cDDP). Activation of the GADD153 promoter coupled to the luciferase gene and transfected into human ovarian carcinoma 2008 cells correlated well with the increase in endogenous GADD153 mRNA after treatment with taxol but not after treatment with cDDP. Following treatment with cDDP, the increase in endogenous GADD153 mRNA was 10-fold greater than the increase in GADD153 promoter activity. Likewise, at equitoxic levels of exposure (IC80), cDDP produced a 5-fold greater increase in endogenous GADD153 mRNA than taxol. The tyrosine kinase inhibitor tyrophostin B46 had no significant effect on the ability of taxol to activate the GADD153 promoter, but inhibited activation of the GADD153 promoter by cDDP in a concentration-dependent manner. Tyrphostin B46 synergistically enhanced the cytotoxicity of cisplatin; however, the same exposure had no significant effect on the cytotoxicity of taxol. We conclude that (1) taxol and cDDP activate GADD153 promoter activity through different mechanisms; (2) the signal transduction pathway mediating induction by cDDP involves a tyrosine kinase inhibitable by tyrphostin B46; and (3) that inhibition of this signal transduction pathway by tyrphostin synergistically enhances cDDP toxicity.

Regulation of initial vinblastine influx by P-glycoprotein.

P-glycoprotein (PGP) is an energy-dependent efflux pump that serves to protect cells against the cytotoxicity of many natural product drugs including vinblastine (VBL). In this study we investigated the role of PGP in regulating initial VBL influx. The apparent influx of VBL, measured over the first 20 s, was 2-fold lower in KB-GRC1 cells expressing a transfected mdr1 gene at high level than in non-expressing parental KB-3-1 cells. Inhibition of PGP efflux function with dipyridamole increased the influx rate constant by 4.0-fold in the KB-GRC1 cells but only 2.1-fold in the KB-3-1 cells. Verapamil, another inhibitor of PGP-mediated efflux, increased the initial influx rate constant by 2.7-fold in the KB-GRC1 cells but only 1.4-fold in the KB-3-1 cells. Inhibition of PGP function by depletion of ATP increased influx by 6.8-fold and 2.2-fold in the two cell types, respectively. Mutation of PGP at both ATP binding sites abolished its ability to limit initial influx. Thus, VBL is serving as an efficient substrate for the efflux pump even within the first few seconds of drug exposure, consistent with the hypothesis that PGP may directly efflux drug from the cell membrane.

Observations on control of N2 and N3 neck disease in squamous cell carcinoma of the head and neck by intra-arterial chemoradiation

Patients with head and neck squamous cell cancer with N2 and N3 neck disease have a poor prognosis and are at risk to fail regionally despite combined surgery and radiation. Twenty‐two patients with N2 and N3 neck disease (and T3‐4 primaries) were treated with intra‐arterial, high‐dose cisplatin (CDDP), 150 mg/m2 per week for 4 weeks, and concurrent radiation. All patients were followed for at least 2 years or until death from any cause. Twenty patients had a complete response at the primary site. Two of the 20 with a complete response later had a neck recurrence and died. Five patients with palpable nodes after treatment underwent fine‐needle aspiration (FNA), one of which was positive and two suggestive of cancer. Six neck dissections were performed in this group, only two of which had positive nodes. This chemoradiation protocol may offer reasonable control of N2 and N3 neck disease in advanced head and neck squamous cell cancer. Neck dissection appeared to be necessary in only those patients with nodes 8 weeks after treatment in whom FNA was positive or suggestive of cancer. Because of the relatively small size of this series, additional accrual and monitoring of such patients is planned.