Synergistic interaction between cisplatin and tamoxifen delays the emergence of cisplatin resistance in head and neck cancer cell lines

The interaction between cisplatin (cDDP) and tamoxifen (TAM) was evaluated in the human head and neck squamous-carcinoma cell lines UM-SCC-10B and UM-SCC-5. Synergy between cDDP and TAM was demonstrated in the UM-SCC-10B cell line. Concordant with the synergistic effect between cDDP and TAM, the rate of development of resistance to cDDP was delayed when selections were performed in the presence of TAM. However, in the UM-SCC-5 cell line, TAM was neither synergistic nor did it delay the development of cDDP resistance. The difference with respect to the synergistic interaction of cDDP with TAM and the effect on the development of cDDP resistance in the UM-SCC-10B and UM-SCC-5 cell lines was not related to any significant difference in the accumulation of the cDDp analog [3H]-cis-dichloro(ethylenediamine)platinum(II) (DEP), drug sensitivity [concentrations inhibiting colony formation by 50% (IC₅₀ values) were 6.5 and 7.2 M for cDDP and 3.5 and 3.2M for TAM, respectively], the number of estrogen and progesterone receptors (negative in both cell lines), the number of antiestrogen binding sites (404±85 and 353±24 fmol/mg protein, respectively), or the affinity of TAM for these binding sites (1.7 and 1.5 nM, respectively). Importantly, however, we demonstrated that TAM can delay the emergence of resistance to cDDP in head and neck carcinomas and that this effect is linked to the nature of the interaction between cDDP and TAM.     

Role of glutathione, glutathione S-transferases and multidrug resistance-related proteins in cisplatin sensitivity of head and neck cancer cell lines.

Resistance to chemotherapy is a major problem in the treatment of patients with head and neck squamous cell carcinoma (HNSCC). Important factors involved are drug detoxification by glutathione (GSH) and reduced drug accumulation due to active transport out of the cell by so-called ''multidrug resistance-related proteins''. We have studied a panel of eight HNSCC cell lines showing differences in sensitivity to the anti-cancer drug cisplatin. Our previous studies indicated that the IC50 values were inversely correlated with the intracellular accumulation of platinum (Pt). In the present study, cellular GSH levels were found not to be related to the IC50 values. The expression levels of the enzymes glutathione S-transferase (GST) alpha, mu, and pi, the multidrug resistance-related proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and the lung resistance protein (LRP) were determined semiquantitatively by means of immunocytochemistry. The levels of the GSTs, P-gp and LRP were not found to be correlated with the IC50 values of the HNSCC cell lines. Surprisingly, however, an inverse correlation was found between MRP levels and IC50 values. The MRP expression levels were in agreement with the results of the MRP functional assay, based on the transport of calcein across the cell membrane as performed for two of the cell lines. Further studies should prove whether other pump mechanisms or DNA repair are involved in the cisplatin accumulation and the subsequent HNSCC cell growth inhibition.     

Butyrate response factor 1 enhances cisplatin sensitivity in human head and neck squamous cell carcinoma cell lines

Cisplatin is a widely used chemotherapeutic agent in head and neck squamous cell carcinoma (HNSCC). Resistance to cisplatin is a common feature of HNSCC. To identify genes that may regulate cisplatin sensitivity, we carried out a cDNA microarray analysis of gene expression in cisplatin-sensitive and cisplatin-resistant HNSCC-derived cell lines. Among genes differentially expressed by cisplatin treatment, we have confirmed the elevated expression of butyrate responsive factor 1 (BRF1) in cisplatin-sensitive HNSCC cells and have demonstrated that the expression level of BRF1 is associated with cisplatin-sensitivity. Specific inhibition of BRF1 expression using an antisense oligodeoxynucleotide (ODN) decreased the cisplatin-sensitivity and, on the contrary, overexpression of BRF1 increased cisplatin-sensitivity in HNSCC cells. Elevated expression of BRF1 decreased the level of the human inhibitor of apoptosis protein-2 (cIAP2) and increased the caspase-3 activity in HNSCC cells. In addition, elevated expression of BRF1 decreased the expression level of enhanced green fluorescent protein (EGFP) linked to a 3' terminal AU-rich element (ARE) of cIAP2 mRNA. These findings demonstrate that BRF1 expression enhanced cisplatin sensitivity in HNSCC cells by reducing the levels of cIAP2 mRNA.     

Relationship between folate-binding protein expression and cisplatin sensitivity in ovarian carcinoma cell lines.

It has been suggested that sensitivity of ovarian carcinomas to cisplatin is in part related to an endogenous folate deficiency. In this work, we investigated whether overexpression of the folate-binding protein (FBP), a receptor involved in folate transport, might be associated with cisplatin sensitivity. The results obtained on a panel of ten ovarian carcinoma cell lines that overexpress different levels of the FBP showed a statistically significant relationship between FBP overexpression and cisplatin responsiveness, with the most sensitive cell lines expressing higher FBP levels on their membrane than the less sensitive ones. The relationship was observed both in cells growing in standard medium-containing high-folate concentrations (2.3 microM) and in cells adapted to growth in low-folate (20 nM) medium. Analysis of two cisplatin-resistant cell lines derived from the cisplatin-sensitive IGROV1 ovarian carcinoma cell line indicated that resistance was associated with a significant decrease in FBP expression. However, the receptor does not appear to be directly responsible for drug sensitivity per se as different cell lines transfected with FBP cDNA did not become more sensitive to the drug. Together, the data suggest the possible predictive value of FBP in ovarian carcinoma, as higher levels of expression can be indirectly but significantly associated with increased drug sensitivity.     

Decreased cisplatin/DNA adduct formation is associated with cisplatin resistance in human head and neck cancer cell lines

Purpose: To evaluate the correlation between cisplatin sensitivity, intracellular glutathione, and platinum/DNA adduct formation (measured by atomic absorption spectroscopy) in a series of seven head and neck cancer cell lines, and to evaluate the effect of biochemical modulation of glutathione on platinum/DNA adduct formation and repair. Methods: Cisplatin/DNA adducts were measured by atomic absorption spectroscopy. Glutathione content was measured by enzymatic assay and was modulated with buthionine sulfoximine. Apoptosis was measured by double-labeled flow cytometry. Results: Intracellular glutathione concentration was strongly correlated with cisplatin resistance (P = 0.002, R ²=0.7). There was also a statistically significant inverse correlation between cisplatin/DNA adduct formation and the IC₅₀ for cisplatin in these cell lines. (P=0.0004, R ²=0.67). In addition, resistant cells were able to repair approximately 70% of cisplatin/DNA adducts at 24 h, while sensitive cells repaired less than 28% of adducts in the same period. However, despite the positive correlation between cellular glutathione and cisplatin resistance, there was no direct correlation between intracellular glutathione concentration and platinum/DNA adduct formation. Further, depletion of intracellular glutathione by buthionine sulfoximine did not dramatically alter formation of cisplatin/DNA adducts even though it resulted in marked increase in cisplatin cytotoxicity and was associated with increased apoptosis. Conclusions: These results suggest that glutathione has multiple effects not directly related to formation of cisplatin/DNA adducts, but may also be an important determinant of the cell's ability to repair cisplatin-induced DNA damage and resist apoptosis. Received: 9 December 1999 / Accepted: 10 May 2000     

Oxaliplatin activity in head and neck cancer cell lines

Oxaliplatin (cis-[(1R,2R)-1,2-cyclohexanediamine-N,N] [oxalato(2-)-O,O] platinum; Eloxatin) is a third-generation platinum compound with a 1,2-diaminocyclohexane (DACH) carrier ligand, which has a wide spectrum of anticancer activity in vitro systems and has displayed preclinical and clinical activity in a wide variety of tumors. To investigate its in vitro activity against head and neck cancer, we exposed two head and neck cancer cell lines to the compound, created a variant resistant to cisplatin to study cross-resistance to the compound and analyzed the potential radiosensitizing effect of the drug. We report here that oxaliplatin was cytotoxic at similar doses to cisplatin in these cells. There was no cross-resistance to cisplatin, as demonstrated by different IC₅₀ values in these cell lines and the sensitivity to oxaliplatin of the cisplatin-resistant cell line. There was an effective radiosensitizer effect of the compound in either cell line. Additional in vitro and in vivo experimentation is warranted in order to support the use of oxaliplatin as a radiosensitizer in head and neck cancer patients.Magali Espinosa and Moises Martinez contributed equally to this paper.     

Pharmacodynamics of cisplatin in human head and neck cancer: correlation between platinum content, DNA adduct levels and drug sensitivity in vitro and in vivo.

Total platinum contents and cisplatin-DNA adduct levels were determined in vivo in xenografted tumour tissues in mice and in vitro in cultured tumour cells of head and neck squamous cell carcinoma (HNSCC), and correlated with sensitivity to cisplatin. In vivo, a panel of five HNSCC tumour lines growing as xenografts in nude mice was used. In vitro, the panel consisted of five HNSCC cell lines, of which four had an in vivo equivalent. Sensitivity to cisplatin varied three- to sevenfold among cell lines and tumours respectively. However, the ranking of the sensitivities of the tumour lines (in vivo), also after reinjection of the cultured tumour cells, did not coincide with that of the corresponding cell lines, which showed that cell culture systems are not representative for the in vivo situation. Both in vitro and in vivo, however, significant correlations were found between total platinum levels, measured by atomic absorption spectrophotometry (AAS), and tumour response to cisplatin therapy at all time points tested. The levels of the two major cisplatin-DNA adduct types were determined by a recently developed and improved 32P post-labelling assay at various time points after cisplatin treatment. Evidence is presented that the platinum-AG adduct, in which platinum is bound to guanine and an adjacent adenine, may be the cytotoxic lesion because a significant correlation was found between the platinum-AG levels and the sensitivities in our panel of HNSCC, in vitro as well as in vivo. This correlation with the platinum-AG levels was established at 1 h (in vitro) and 3 h (in vivo) after the start of the cisplatin treatment, which emphasizes the importance of early sampling.     

Inhibition of cytotoxicity of cisplatin by cyclooxygenase-2 inhibitor nimesulide in head and neck cancer cell lines

Although head and neck cancer is a common malignancy, investigations have not yet improved the poor prognosis of patients. Therefore, it is important to find new cancer treatment modalities. Recent studies showed that cyclooxygenase, especially its isoform cyclooxygenase-2, is involved in tumorigenesis, tumor growth and metastasis. Inhibition of this enzyme by cyclooxygenase inhibitors has been shown to be antiproliferative in numerous cancer cell lines. The aim of this study was to investigate if the selective cyclooxygenase-2 inhibitor nimesulide could enhance cytotoxicity of the standard chemotherapeutic agent cisplatin. Head and neck squamous cell cancer cells were incubated with nimesulide and/or cisplatin, and counted after 24, 48 and 72 h treatment. Visualization of apoptotic cells was done by immunohistochemistry. We demonstrated that nimesulide inhibits the cytotoxic effect of cisplatin in HNSCC cells. Therefore, COX-2 inhibitor nimesulide may not be a good partner for cisplatin in combination therapy for cancer.     

Morphological change and cellular differentiation induced by cisplatin in human neuroblastoma cell lines

Summary In 1986 we reported on the capacity ofcis-diamminedichloroplatinum(II) (cisplatin, CDDP) to induce erythroid cellular differentiation in the K562 cell [9]. To continue our study of the differentiating activity of cisplatin, we treated two human neuroblastoma cell lines with different doses of the drug in vitro. Both cell lines showed changes in morphology; however, only one achieved a fully differentiated neuronal phenotype (cisplatin concentration 1 g/ml). The differentiated neuroblastoma cells exhibited extensive neurite outgrowth that reached maximal elongation after 5 days of culture, forming several interconnections. Cisplatin could induce neuronal differentiation, as did retinoic acid, a neuroblastoma-differentiating agent. The results show that cisplatin should be a candidate for further in vitro and in vivo studies of induced differentiation.     

Characterisation of the P53 status,BCL-2 expression and radiation and platinum drug sensitivity of a panel of human ovarian cancer cell lines

The P53gene is frequently mutated in late stage ovarian cancer and has been proposed as a determinant of radiation and chemosensitivity. We have therefore determined the p53 functional status, P53sequence, radiation sensitivity and cytotoxicity of cisplatin and the novel platinum analogue, AMD473, in a panel of 6 human ovarian cancer cell lines. Constitutive p53 protein levels were low in A2780, CH1, LK1, LK2 and PA1 but were markedly induced following irradiation. In OV1P, constitutive p53 protein was readily detectable and levels were induced slightly following irradiation. P21^WAF1/CIP1 and MDM-2 mRNA were constitutively expressed in all the cell lines and expression was induced markedly following irradiation. There was marked radiation induced G₁/S arrest in A2780 but only partial arrests in CH1, LK1, LK2, PA1 and OV1P lines. No mutations were found in A2780, CH1, LK1, LK2 and PA1 cells by single-strand conformational polymorphism (SSCP) analysis but a heterozygous point mutation was found in exon 5 of OV1P. All the cell lines were radiation sensitive and also relatively sensitive to cisplatin; however, OV1P was the most resistant being consistent with its heterozygous P53 status. AMD473 was less potent than cisplatin but a similar pattern of drug sensitivity was observed with the exception of LK2, which was resistant. CH1, LK1, LK2 and PA1 all expressed BCL-2 protein but there was no expression in A2780 and OV1P. Our results suggest an overall association between wild type P53 and radiation and platinum drug sensitivity in these ovarian cancer cell lines. Int. J. Cancer 77:913–918, 1998.© 1998 Wiley-Liss, Inc.     

High-dose cisplatin in advanced head and neck cancer

Summary In 22 patients with advanced squamous cell carcinoma of the head and neck we evaluated the efficacy and toxicity of 200 mg/m² cisplatin administered in 3% NaCl with vigorous hydration. Six patients had previously untreated stage IV disease and 16 patients had recurrent disease, including eight with prior chemotherapy including low-dose cisplatin and carboplatin. Cisplatin was administered as a brief infusion, either 40 mg/m²/day × 5 or 50mg/m²/day × 4, every 28 days. Objective responses were observed in 16 of 22 (73%) patients, including 5 of 6 (83%) previously untreated patients and 11 of 16 (69%) patients with recurrent disease. This included two comoplete responses, one confirmed pathologically. Fifty-seven courses of drug were administered and toxicity was monitored with serial creatinine clearance determinations, audiograms, and sensorimotor exams. Neuropathy and ototoxicity were dose-limiting and led to the stopping of treatment in 12 of the 16 responders after one to four courses (median three courses). Only two responding patients continued treatment until disease progression occurred at 3 and 4 months after achieving maximum response. Acute, transient nephrotoxicity occurred in four patients; two were retreated. Moderate myelosuppression occurred in all patients but was not treatment-limiting. For most patients the maximally tolerated number of courses was three. The median survival time was 33.5 weeks for recurrent disease patients, 108 weeks for newly diagnosed patients. This regimen is not recommended for the palliation of recurrent disease. However, the very high response rate suggests that high-dose cisplatin may have a useful role in induction or adjuvant chemotherapy regimens.Supported in part by Public Health Service Grant 5-MO1-RR-00042, from the National Institutes of Health and Human ServicesPresented in part at the 21st annual meeting of the American Society for Clinical Oncology, Houston, Texas, May 19–21, 1985     

KiSS1 mediates platinum sensitivity and metastasis suppression in head and neck squamous cell carcinoma

Although surgery and radiotherapy have been the standard treatment modalities for head and neck squamous cell carcinoma (HNSCC), the integration of cisplatin (CDDP)-based therapy has led to improvements in local and regional control of disease for patients. However, many trials show that only 10-20% of patients benefit from this treatment intensification, which can result in profound treatment-associated morbidity and mortality. Moreover, the marginal survival improvement suggests that CDDP resistance is an innate characteristic of HNSCC. To elucidate the biological mechanisms underpinning CDDP resistance in HNSCC, we utilized an experimental model of CDDP resistance in this disease. We first observed significant enhancements in local tumor growth and metastasis, as well as adverse survival, in CDDP-resistant (CR) tumors compared with sensitive tumors. To elucidate the molecular mechanisms of this phenotype, we undertook a systems biology-based approach utilizing high-throughput PCR arrays, and we identified a significant suppression of KiSS1 mRNA and protein expression in the CR cells, but no significant regions of genomic loss with array comparative genomic hybridization. Genetic suppression of KiSS1 in CDDP-sensitive cell lines rendered them CR, an observation that was mechanistically linked to alterations in glutathione S-transferase-π expression and function. We next confirmed that, in human HNSCC tumors, loss of KiSS1 expression was associated with metastatic human HNSCC tumors compared with non-metastatic tumors. Genetic reconstitution of KiSS1 in CR cells abrogated cellular migration and induced CDDP sensitivity. To confirm these findings in a murine model, either CR or KiSS1-transfected CR cells were studied in an orthotopic model of HNSCC, or survival studies revealed significant improvement in survival of the mice bearing CR-KiSS1 tumors. Mechanistically, alterations in apoptotic pathways and CDDP metabolism contributed to KiSS1-associated chemotherapy sensitization. These studies provided further direct evidence for the role of KiSS1 loss in biologically aggressive HNSCC and suggest potential targets for therapy in CR cancers.     

Interaction between potential doubling time and TP53 mutation: predicting radiotherapy outcome in squamous cell carcinoma of the head and neck

PURPOSE: To investigate the correlation between tumor potential doubling time, Tpot, and mutations in the p53 gene, TP53, and the potential of these parameters to predict outcome of head and neck cancer patients treated with radiotherapy. METHODS AND MATERIALS: Data from two independent studies on Tpot and TP53 mutations were combined, including 58 patients with squamous cell carcinoma of the head and neck. Tpot was estimated on biopsies obtained 6-9 h after infusion of iododeoxyuridine by combined flow cytometry and immunohistology. TP53 mutations were detected using DGGE and sequenced. All patients received primary radiotherapy alone. RESULTS: The predictive value of Tpot alone was of borderline significance. However, in TP53 wild-type tumors, Tpot was a strong predictor of outcome, whereas Tpot in TP53 mutant tumors failed to provide any information. Tpot and TP53 were not associated with nodal control; however, there was a strong relationship with control in the T-position, disease-specific survival, and overall survival. CONCLUSION: Tpot can to be a relevant parameter for predicting outcome of radiotherapy in head and neck cancer but only in the subset of patients without mutations in the p53 gene.     

Polymorphism of FGFR4 in cancer development and sensitivity to cisplatin and radiation in head and neck cancer

The aim of this study was to investigate the predisposition of the FGFR4 Gly/Arg polymorphism for development of head and neck squamous cell carcinoma (HNSCC) and, furthermore, to examine if the FGFR4 Arg(388) allele can be associated with resistance to chemo- and radiotherapy. When analysing 110 tumour biopsies a significant 1.7-fold increased risk to develop HNSCC in individuals carrying the Gly(388) allele (p=0.026) was found. Moreover a 2-fold increased risk for males harbouring the Gly(388) allele (p=0.031) to develop HNSCC was detected. In 39 HNSCC cell lines the role of the Arg(388) allele for radiation and cisplatin sensitivity was investigated. Our results show no role of the Arg(388) allele for the radiosensitivity (p=0.996) but indicate a tendency to increased cisplatin sensitivity (p=0.141). When screening the transmembrane and kinase domains in the FGFR4 gene a novel mutation, probably generating a truncated protein lacking exons 14-18, was found in six of eight selected cell lines. Taken together, we have here identified a marker that predicts the risk to develop HNSCC and possibly the sensitivity to cisplatin as well as a novel mutation in the FGFR4 gene.     

Pharmacokinetic evaluation of platinum derived from cisplatin administered alone and with pemetrexed in head and neck cancer patients

Purpose  This phase I study characterized the pharmacokinetics of free and total platinum derived from cisplatin administered alone and in combination with pemetrexed. Secondary objectives were to assess the pharmacokinetics of pemetrexed when it is combined with cisplatin as well as to evaluate the safety profile and document antitumor activity associated with this combination. Methods  An open-label, two-arm, cross-over phase 1 study was performed in patients with squamous cell carcinoma of the head and neck, age ≥18 years, an Eastern Cooperative Oncology Group performance status of 0–2, and adequate organ function. Blood samples were taken and pharmacokinetics evaluated for the first two cycles using noncompartmental analysis. Patients received either pemetrexed (500 mg m⁻²) plus cisplatin (75 mg m⁻²) administered in cycle 1 followed by cisplatin alone in cycle 2; or in the reverse order (i.e., cisplatin alone in cycle 1 followed by pemetrexed plus cisplatin in cycle 2). Each treatment cycle was 21 days and patients received folic acid, vitamin B₁₂ supplementation, and dexamethasone prophylaxis. After the first two cycles, patients continued study treatment with pemetrexed plus cisplatin every 3 weeks up to a maximum of six total treatment cycles. Toxicities were graded by the investigators according to the National Cancer Institute Common Toxicity Criteria for Adverse Events (CTCAE), version 3.0. Results  A total of 13 patients were treated; one patient was discontinued from the study after cycle 1 for failure to meet baseline eligibility criteria for renal function. The ratios and 90% confidence intervals (CI) comparing the pharmacokinetics for cisplatin administered with pemetrexed to those for cisplatin administered alone for free platinum were: C _max = 1.08 (CI: 0.92, 1.27) and AUC = 0.93 (CI: 0.82, 1.06); and, total platinum were: C _max = 0.97 (CI: 0.88, 1.06) and AUC = 0.87 (CI: 0.81, 0.93). These results indicate that platinum pharmacokinetics (free and total) are similar, whether cisplatin is administered alone or combined with pemetrexed. The pemetrexed pharmacokinetic results were consistent with those from previous single-agent pemetrexed studies and a previous study of pemetrexed in combination with cisplatin. The combination of pemetrexed and cisplatin did not show any unexpected toxicities. Consistent with the platinum pharmacokinetic results, co-administration with pemetrexed did not appear to enhance cisplatin-related toxicities. Of the 13 treated patients, 11 had stable disease as the best overall response and 2 had progressive disease. Conclusions  The pharmacokinetics of free platinum derived from cisplatin were not altered by co-administration with pemetrexed, and in agreement with this, no unexpected cisplatin-induced toxicities were observed when these drugs were combined.