The Synergistic Cytotoxicity of Cisplatin and Taxol in Killing Oral ...

Jpn J Clin Oncol 2004;34(9)499–504 doi:10.1093/jjco/hyh091

Original Articles

The Synergistic Cytotoxicity of Cisplatin and Taxol in Killing Oral Squamous Cell Carcinoma Guan-Cheng Huang1, Shyun-Yeu Liu2, Mei-Huei Lin3, Yung-Yen Kuo2 and Young-Chau Liu4 1

Department of Internal Medicine and 2Oral and Maxillofacial Surgery Section, Chi-Mei Medical Center, Tainan, Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan and 4College of Liberal Education, Shu-Te University, Kaohsiung County, Taiwan, Republic of China

3

Received April 12, 2004; accepted July 3, 2004

Background: Platinum, 5-fluorouracil (5-FU) and taxanes are commonly used in chemotherapeutic modalities of various carcinomas. However, taxanes are rarely used in patients suffering from head and neck squamous cell carcinoma (HNSCC) in Taiwan. The purpose of this study was to assess whether there is a synergistic effect produced by incorporating Taxol (paclitaxel) with cisplatin, carboplatin or 5-FU in the combined treatment of oral squamous cell carcinoma (OSCC). Methods: OSCC cells were surgically excised from a Taiwanese patient and cultured into a cell line, OECM-1. The viability of OECM-1 after drug treatment was determined by an XTT labeling reagent. Results: The dose-dependent cytotoxicity of each drug was determined. The order of chemosensitivity of OECM-1 toward these drugs was Taxol, cisplatin, carboplatin and 5-FU, with 50% inhibitory concentrations (IC50s) of 10, 68, 332 and 3000 mM, respectively. In the combined drug treatment, low concentrations of platinum (10 mM) or 5-FU (500 mM) were included in the culture media with low cytotoxic concentrations of Taxol (0.025, 0.05 and 0.1 mM). When combined with 0.025 mM of Taxol, only cisplatin, rather than carboplatin and 5-FU, showed synergistic cytotoxicity with OECM-1. Cisplatin also acted synergistically with 0.05 and 0.1 mM of Taxol. On the other hand, carboplatin and 5-FU acted additively with low cytotoxic concentrations of Taxol (0.025, 0.05 and 0.1 mM). Conclusions: Our preliminary results suggest that there may be a beneficial outcome in incorporating Taxol into the chemotherapeutic modalities of HNSCC patients in Taiwan. Furthermore, at least some of the OSCC cells may be more sensitive to Taxol/cisplatin than to Taxol/carboplatin or Taxol/5-FU treatment. Key words: carboplatin – cisplatin – 5-fluorouracil – oral squamous cell carcinoma – Taxol

INTRODUCTION Taxanes, including Taxol (paclitaxel) and Taxotere (docetaxel), have been known to block cell replication, arrest cells in the G2 and M phase of the cell cycle, and stabilize cytoplasmic microtubules, for more than two decades (1). They have not only demonstrated a unique ability to palliate the symptoms of many types of advanced cancers, including carcinomas of the ovary, lung, head and neck, bladder and esophagus, but they have also demonstrated effectiveness in the

For reprints and all correspondence: Young-Chau Liu, No. 59, Hun Shan Road, Yen Chau 824, College of Liberal Education, Shu-Te University, Kaohsiung County, Taiwan, Republic of China. E-mail: [email protected]

initial therapy of earlier stage cancers, a setting in which any new therapy is likely to make its greatest impact (2). To improve the prognostic response, efforts are being put into searching for regimens combined with taxanes. Recently, the combination of Taxol and mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor has been verified to modulate similar genes in lung, ovarian and melanoma cancer cells, and may represent a rational new approach to chemotherapy (3). For more than a decade, the de facto ‘standard of care’ for the palliative management of recurrent head and neck squamous cell carcinoma (HNSCC) has been the combination of platinum (including cisplatin and carboplatin) and 5-fluorouracil (5-FU) (4). Combinations of taxanes with these drugs are now being used as a new first-line chemotherapy in advanced # 2004 Foundation for Promotion of Cancer Research

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Chemosensitivity of oral cancer cells

ovarian cancers (5,6). When combined with radiotherapy, docetaxel exhibits radiation-sensitizing activity against nonsmall-cell lung cancer, esophageal cancer, and HNSCC, offering survival benefits by improving locoregional control and treating micrometastatic disease (7). Taxanes and platinumcombined regimens are also concurrently being used in the treatment of HNSCC, with promising results in countries other than Taiwan (8–10). Furthermore, Taxol, ifosfamide and carboplatin-induced chemotherapy is associated with a high complete response rate at the primary sites, and has had excellent survival and organ preservation rates in a phase II study at the University of Texas M. D. Anderson Cancer Center, Houston, TX. This regimen is also well tolerated in the majority of the patients treated (11,12). Oral squamous cell carcinoma (OSCC) is categorized as a particular type of HNSCC. Traditionally, OSCC patients in Taiwan have been commonly treated with cisplatin (or carboplatin) and/or 5-FU, as in other countries (13). However, the literature specifically describing the combined effects of Taxol with other agents or radiation on oral cancers is accumulating and promising (14–16). To analyze the potential application of Taxol in the treatment of OSCC in Taiwan, we have tested the individual dose-dependent cytotoxicity, 50% inhibitory concentrations (IC50s), and the combined cytotoxic effects of Taxol, cisplatin, carboplatin and 5-FU on an OSCC cell line, OECM-1, derived from a Taiwanese patient (17).

MATERIALS AND METHODS CELLS AND CULTURE CONDITIONS Human oral squamous cell carcinoma (OSCC) cell line, OECM-1, was kindly provided by Dr Kuo-Wei Chang (Institute of Oral Biology, National Yang-Ming University). The cultivation conditions have been described in our previous report (18). Briefly, OECM-1 is routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Paisley, UK) containing 10% fetal bovine serum (FBS) (HyClone, Vermont, USA), at 37 C in a humid atmosphere with 5% CO2. CELL VIABILITY ASSAY OECM-1 was seeded in a 96-well plate, at a density of 3000 cells/ well in 0.2 ml DMEM containing 10% FBS. After overnight incubation under the same cultivating conditions, each well was refreshed with 0.2 ml serum-free medium (SFM) for another day. Cells were then treated with 0.2 ml SFM containing various concentrations of Taxol, cisplatin, carboplatin, 5-FU, Taxol/ cisplatin, Taxol/carboplatin or Taxol/5-FU. Because the standard powder of docetaxel was not available, Taxol was the only taxane tested in this study. The drug-containing SFM was refreshed after 2 days, and incubated under the same conditions for another 2 days. Finally, cell viability was accessed with an XTT reagent (Sigma Diagnostics, Inc., St Louis, MO), and by measuring the absorbance at 450 nm with a plate reader. Relative

viability was obtained from the absorbance at 450 nm of drugtreated OECM-1 cells/the absorbance at 450 nm of untreated OECM-1 cells. The same experiment was repeated three times with similar results. ADDITIVE AND SYNERGISTIC CYTOTOXICITY ANALYSIS We used the combination index method of Chou and Talaly (19) to determine whether the observed interactions between Taxol and the other three drugs were additive or synergistic. If the interaction was additive, the sum of the effects of the two drugs should have been equal to the product of their fractional activities. The representative function is: f(u)1,2 = f(u)1 · f(u)2; where f(u)1 = the fraction unaffected by drug 1, f(u)2 = the fraction unaffected by drug 2 and f(u)1,2 = the fraction unaffected by drugs 1 and 2. The expected (presumed to be additive) and observed survival rates of OECM-1 obtained from the three independent drug-combined treatments were analyzed by Student’s t-test. P < 0.05 was viewed as significant.

RESULTS TITRATION OF CELL NUMBERS IN THE CELL VIABILITY ASSAY The XTT (sodium 30 -[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) labeling reagent was originally described for use in the measurement of the drug sensitivity of tumor cell lines (20). The more viable cells generate more orange formazan dye (maximal absorbance at 450 nm wavelength) from the yellow tetrazolium salt of the XTT. Therefore, the absorbance at 450 nm is proportional to the cell number and viability. We tested and verified that the absorbance at 450 nm was linearly proportional from 500 to 5000 OECM-1 cells in a single well of the 96-well culture plate (data not shown). DOSE-DEPENDENT CYTOTOXICITY OF TAXOL, CISPLATIN, CARBOPLATIN AND 5-FU OECM-1 was seeded into a 96-well plate at a density of 3000 cells/well and treated with various doses of Taxol, cisplatin, carboplatin and 5-FU. After 4 days of drug treatment, the viability of the cells was identically assessed with the XTT reagent, as described above. One of three independent results was shown, and all of these four drugs showed dose-dependent cytotoxicity on OECM-1 (Fig. 1). However, in terms of drug concentration, OECM-1 was most sensitive to Taxol treatment (Fig. 1A). In this experiment, the IC50s of Taxol, cisplatin, carboplatin and 5-FU were tested to be 10, 68, 332 and 3000 mM, respectively (Fig. 1). THE COMBINED CYTOTOXIC EFFECT OF TAXOL WITH PLATINUM OR 5-FU The responses of OECM-1 were analyzed with the treatment of low cytotoxic concentrations of Taxol (0.025, 0.05 and

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Figure 1. Dose-dependent cytotoxicity of Taxol, cisplatin, carboplatin and 5-fluorouracil (5-FU). Relative viability was obtained from the absorbance at 450 nm of drug-treated OECM-1 cells/the absorbance at 450 nm of untreated OECM-1 cells. Data are presented as mean – SD (error bar).

0.1 mM) with cisplatin (10 mM), carboplatin (10 mM) or 5-FU (500 mM). Each of the three experiments is shown in Fig. 2. Of the three different combinations (Taxol/cisplatin, Taxol/ carboplatin and Taxol/5-FU), Taxol/cisplatin (Fig. 2A) exhibited the most dramatic reduction of cell viability, followed by Taxol/carboplatin (Fig. 2B) and Taxol/5-FU (Fig. 2C). To further evaluate whether cisplatin, carboplatin or 5-FU acted additively or synergistically with Taxol in killing OECM-1, the observed cell survival rate after cisplatin/ Taxol, carboplatin/Taxol or 5-FU/Taxol treatment was compared to the predicted survival rate (if these two drugs acted additively) as described in Materials and Methods. Table 1 shows that after cisplatin (10 mM)/Taxol (0.025, 0.05 and 0.1 mM) treatment, the average observed survival rate was significantly lower than that of the predicted survival rate (P < 0.05). Therefore, cisplatin may act synergistically with Taxol. On the other hand, the observed survival rates of carboplatin/Taxol (Table 2) or 5-FU/Taxol (Table 3) combined treatments were similar to the predicted survival rates. These results suggest that carboplatin and 5-FU may act additively with Taxol.

DISCUSSION The treatment of oral cavity and oropharyngeal squamous cell carcinomas shows a tendency to involve non-surgical modalities (21). With head and neck cancer in aged and often emaciated patients, the reduction of the side effects of any modality is a major task in order to secure the success of a multimodality regimen. On the other hand, intensification of the treatment protocol should be pushed to extremes, either by dense interlacing of modalities or by a combination of as many modalities as possible to enhance the efficacy of treatment by synergistic effects (16,21). Emerging evidence suggests the benefits of Taxol in the treatment of oral carcinomas. For example, the well-known regional effectiveness of radiation can be enhanced by the accompanying radiosensitizing effects of chemotherapy, for which the taxanes promise to be a suitable agent (22,23). Eckardt et al. (14) demonstrated impressive clinical and pathological response rates for concurrent taxol/carboplatin and radiotherapy as a preoperative treatment modality in advanced oral and oropharyngeal cancer. Kovacs et al. (16) also proved

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Table 1. Analysis of cisplatin/Taxol-combined effects on the viability of OECM-1 cells Combination (mM)

Cisplatin (10) + Taxol (0.025)

Survival rate Expected

Observed

0.95 · 0.98 = 0.94

0.56 (1st experiment)

0.81 · 0.92 = 0.75

0.51 (2nd experiment)

0.88 · 0.89 = 0.78

0.45 (3rd experiment)

Average survival rate (mean – SD)

0.82 – 0.10

0.51 – 0.06 (P = 0.009)


0.95 · 0.80 = 0.76


0.81 · 0.73 = 0.59


0.88 · 0.82 = 0.72



0.69 – 0.09

0.42 – 0.05 (P = 0.009)


0.95 · 0.75 = 0.71


0.81 · 0.65 = 0.53


0.88 · 0.69 = 0.61


0.62 – 0.09

0.39 – 0.06 (P = 0.014)


This table presents a comparison of the predicted viability (based on an assumption that the drug-combined activities are additive) and the experimental viability after treating OECM-1 with cisplatin (10 mM) plus various concentrations of Taxol (0.025, 0.05 or 0.1 mM). Calculation of the predicted survival rate is described in Materials and Methods. Values of the predicted and observed survival rates from three independent experiments were statistically analyzed with a t-test. Table 2. Analysis of carboplatin/Taxol-combined effects on the viability of OECM-1 cells Combination (mM)

Carboplatin (10) + Taxol (0.025)

Figure 2. Combined drug effects on the viability of OECM-1. (A) Taxol (0, 0.025, 0.05, 0.1 mM)/cisplatin (10 mM). (B) Taxol (0, 0.025, 0.05, 0.1 mM)/ carboplatin (10 mM). (C) Taxol (0, 0.025, 0.05, 0.1 mM)/5-fluorouracil (5-FU) (500 mM).

that radical neck dissection (nodal involvement), followed by adjuvant chemoradiation over 5 weeks (51.9 Gy, systemic docetaxel 25 mg/m2, once every week) was feasible, and showed better survival for the whole population and for all tumor stages. It has also been suggested that the incorporation of docetaxel in chemoradiotherapy regimens for the treatment


Observed

0.90 · 0.98 = 0.88


0.78 · 0.92 = 0.72


0.55 · 0.89 = 0.49



0.70 – 0.20

0.85 – 0.11 (P = 0.163)


0.90 · 0.80 = 0.72


0.78 · 0.73 = 0.57


0.55 · 0.82 = 0.45



0.58 – 0.14

0.54 – 0.17 (P = 0.383)


0.90 · 0.75 = 0.68


0.78 · 0.65 = 0.51


0.55 · 0.69 = 0.38


0.52 – 0.15

0.55 – 0.23 (P = 0.437)


The data presented in this table represent the same analyses as Table 1, except that carboplatin is substituted for cisplatin.

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Table 3. Analysis of 5-fluorouracil (5-FU)/Taxol-combined effects on the viability of OECM-1 cells Combination (mM)

5-FU (500) + Taxol (0.025)


Observed

0.88 · 0.98 = 0.86


0.80 · 0.92 = 0.74


0.92 · 0.89 = 0.82



0.81 – 0.06

0.76 – 0.14 (P = 0.319)

5-FU (500) + Taxol (0.05)

0.88 · 0.80 = 0.70


0.80 · 0.73 = 0.58


0.92 · 0.82 = 0.75



0.67 – 0.09

0.57 – 0.11 (P = 0.122)

5-FU (500) + Taxol (0.1)

0.88 · 0.75 = 0.66


0.80 · 0.65 = 0.52


0.92 · 0.69 = 0.63


0.60 – 0.07

0.56 – 0.15 (P = 0.351)


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It has been demonstrated by Kano et al. that the interaction of Taxol and cisplatin may be schedule- and cell line-dependent (26). The optimal schedule of this combination may be Taxol first followed by cisplatin. In our present study, the 4-day simultaneous treatment of OECM-1 with Taxol and cisplatin showed synergistic rather than protective effects. However, the possibility of achieving more promising cytotoxicity by the scheduled drug treatment is not ruled out and is being investigated. It is also possible that the drug response of different HNSCC cells may fluctuate case by case. This study, however, has strengthened the evidence for the benefits of Taxol in treating HNSCC patients in Taiwan. The unraveling of the underlying mechanisms may provide an important diagnostic reference in the future, which can be used for making decisions on the most effective chemotherapy regimens.

Acknowledgements We would like to thank Dr Yulun Huang (Chi-Mei Medical Center) for providing us with Taxol (paclitaxel), 5-FU, cisplatin and carboplatin. This study was supported by the Chi-Mei Medical Center, project number CMFHR9250.

The data presented in this table represent the same analyses as Table 1, except that 5-FU is substituted for cisplatin.

References of HNSCC adds incrementally to the efficacy of cisplatin and 5-FU (8,9). Despite these studies, patients with HNSCC have rarely been treated with taxanes in Taiwan. By studying the drug response of an OSCC cell line (OECM-1) derived from a male Taiwanese, we were able to show that these cells were most sensitive to Taxol treatment (IC50 = 10 mM) as compared to platinum and 5-FU (Fig. 1). By using single-agent treatment, two Phase II trials have shown modest activity (overall response rates of 40% and 35%) in recurrent or metastatic HNSCC patients (24,25). Thus, together with our in vitro analysis, Taxol may be a promising agent for HNSCC therapy. In addition, we have also shown that cisplatin exhibited synergistic cytotoxicity on OECM-1 with low cytotoxic concentrations of Taxol (Fig. 2 and Table 1), while carboplatin and 5-FU acted additively with low cytotoxic concentrations of Taxol (Fig. 2, Table 2 and Table 3). Although more studies are required, these preliminary results suggest that a better therapeutic response from HNSCC or OSCC patients can be expected in Taiwan, if Taxol is included in the chemotherapy regimens. This speculation is further supported by the recent observation that an excellent long-term survival rate was achieved by incorporating docetaxel into cisplatin and 5-FU combined chemotherapy (8). Although carboplatin is thought to be a better choice than cisplatin in treating ovary and lung cancer patients, we have, for the first time, provided in vitro evidence that some, if not most, OSCC cells may preferentially respond to cisplatin/ Taxol-combined treatment.

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