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Year : 2015  |  Volume : 11  |  Issue : 1  |  Page : 37-40

The effects of combination of gefitinib and cisplatin on tongue squamous cell carcinoma cell lines

1 Department of Oral and Maxillofacial Surgery, Jiamusi University Second Affiliated Hospital, Jiamusi, Heilongjiang Province, China
2 Department of Dental Implantology, Jiamusi University Second Affiliated Hospital, Jiamusi, Heilongjiang Province, China

Date of Web Publication16-Apr-2015

Correspondence Address:
De Chao Li
Department of Dental Implantlogy, Jiamusi University Second Affiliated Hospital, No. 522, Hongqi Street, Jiamusi, Heilongjiang Province
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.147380

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 > Abstract 

Objectives: To study the inhibitory effect of combining the gefitinib with cisplatin on tongue squamous cell carcinoma cells.
Materials and Methods: Two cell lines were used, tca8113 which is a cisplatin-sensitive cell line, and tca8113/CDDP which is a cisplatin-resistant cell line established in Jiamusi University Laboratories. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to calculate the IC 50 values of both drugs using both cell lines. Detection of Ras, Raf and ERK1/2 was done by using western blot technique after exposure to different concentrations of gefitinib and cisplatin.
Results: Tca8113/CDDP has shown more resistance to cisplatin than tca8113 and slightly more resistance to gefitinib than tca8113. Combination of the two drugs has shown to be more effective for both cell lines than when each alone even with lower concentrations.
Conclusions: Combination of gefitinib and cisplatin is a better choice than using each drug alone.

Keywords: Cisplatin, cisplatin resistance, epidermal growth factor receptor, gefitinib, oral cancer

How to cite this article:
Helal HH, Qi CE, Zhao YY, Yao CS, Li DC. The effects of combination of gefitinib and cisplatin on tongue squamous cell carcinoma cell lines. J Can Res Ther 2015;11:37-40

How to cite this URL:
Helal HH, Qi CE, Zhao YY, Yao CS, Li DC. The effects of combination of gefitinib and cisplatin on tongue squamous cell carcinoma cell lines. J Can Res Ther [serial online] 2015 [cited 2020 Feb 22];11:37-40. Available from: http://www.cancerjournal.net/text.asp?2015/11/1/37/147380

 > Introduction Top

Oral and pharyngeal cancer is one of the most common cancers internationally, with around 300,000 new cases each year in the US. [1] Despite the fact that this type of cancer is being studied worldwide, oral cancer survival rate defers according to the location and stage of the cancer. [2] Researchers have focused on the biology of cancer that demonstrated the overexpression of growth receptors and growth factors. Epidermal growth factor receptor (EGFR) is a large family of tyrosine kinases receptors, which is expressed in many cancers including oral cancer. EGFR is responsible for cell proliferation, growth, apoptosis, migration and adhesion. [3] Activation of the EGFR will start the Ras-Raf-MEK-ERK pathway on which we focus our study. [4] A tyrosine kinase inhibitor "gefitinib" is known to inhibit the expression of the EGFR. This attracted many researchers to study the effect of gefitinib on cancer cells. [5] Cisplatin is an anticancer drug which has shown significant effects on oral cancer. Unfortunately, its effects are limited because of the acquired or the intrinsic resistance. Increased expression of EGFR is reported in the cisplatin-resistant cells compared with the cisplatin-sensitive, [6],[7] in this " in vitro" study, we compared the effect of gefitinib and cisplatin, each alone and combined using two cell lines, one is cisplatin-resistant "tca8113/CDDD" tongue squamous cell carcinoma and the other is cisplatin-sensitive "tca8113". This was achieved by measuring the inhibitory rate of both drugs on both cell lines by using 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, then comparing the effect of drugs by detecting Ras, Raf and p-ERK1/2 proteins using western blot analysis.

 > Materials and methods Top

Cell culture

In this study, two cell lines were used, tca8113 a tongue squamous cell carcinoma, and a cisplatin-resistant tca8113/CDDP cell line established in Jiamusi University Laboratories. This was performed by gradual and intermittent exposure to cisplatin, tca8113/CDDP. Cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Thermo Fisher Scientific Inc. UK) with 10% Fetal Bovine Serum (Gibco, Life Technologies, USA) and 100 units/ml penicillin with 100 ug/ml streptomycin and maintained at 37°C and 5% CO 2 .

3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay

Both cell types were seeded in 96-wells plates, at a density of 5000 cells/well and were left overnight to attach. Cells were exposed to different concentrations of gefitinib and cisplatin each alone for 24 h in DMEM-containing drug. For each concentration, six wells were used. 5 mg/ml of Water soluble tetrazolium bromide MTT was added to the wells (50 ul/well) and incubated in dark for 4 h, the supernatant was discarded and the purple crystals were dissolved in DMSO (200 ul/well), absorbance was recorded at 490 nm by spectrophotometer machine (BIO-RAD Laboratories, USA). 50% inhibitory concentration (IC 50 ) "the concentration that caused the death of 50% of cells" was calculated. This was repeated three times in separate occasions.

Western blot assay

Cells were cultured in 10 cm dishes, gefitinib and cisplatin were added to both cell types together with different concentrations as shown in [Figure 1] for 24 h, Culture medium containing drugs was removed; dishes were washed with ice-cold PBS (Solarbio, Beijing, China) twice, Adherent cells were scraped then transferred into precooled tubes. Tubes were centrifuged at 1000 rpm for 5 min; PBS was then removed from the tubes. 4 ul protease and 4 ul phosphatase were dissolved in 100 ul of radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 10 mM NP-40, 5 mM deoxycholic acid, 1 mM sodium dodecyl sulfate, 1 mM ethylenediaminetetraacetic acid, and 1 mM phenylmethylsulfonyl fluoride) was added to each tube, Tubes were then kept for 30 min in 4 degrees refrigerator, and centrifuged at 12,000 rpm for 20 min, The supernatants were transferred into new precooled tubes and proteins concentrations were estimated by using BCA assay, 50ug of proteins were loaded in 10% poly-acryl amide gels which were transferred to PVDF membranes, Membranes were blocked in tris-buffered saline (50 mM Tris-HCl, pH 7.6, and 150 mM sodium chloride) containing 5% nonfat milk and 0.1% Tween 20 (Beyotime Institute of Biotechnology, China) for 1 h at room temperature with agitation, membranes were washed with tris buffered saline with tween (TBST) 4 times 5 min each, They were then cut into pieces, each piece contains a specific protein according to its kDa-so that primary antibodies would be added to the concomitant piece at the same time for all primary antibodies, membranes were incubated with the primary antibody for 24 h in 4degrees refrigerator, antibodies used are: Anti p-ERK1/2, anti pan Ras and anti Raf-1 (Santa Cruz Biotechnology, California, USA), anti B-actin (Wuhan Boster Biological Technology, China), membranes were washed with TBST for 4 times 5 min each, then incubated with secondary antibody "HRP conjugated goat antimouse immunoglobulin G" (Wuhan Boster Biological Technology, China) for 1 h at room temperature. Then washed for 2 times 5 min each with TBST, Protein bands were detected using ECL solution (Wuhan Boster Biological Technology, China) and photographed by X-ray films, which were photographed using v3 western workflow machine (Bio-Rad, USA).
Figure 1: Dishes were categorized as follows: A1-blank, a2-35 uM gefitinib, a3-1.3 ug/ml cisplatin, a4-10 uM of gefitinib + 1 ug/ml cisplatin, a5-20 uM of gefitinib + 1 ug/ml of cisplatin, a6-30 uM of gefitinib + 1 ug/ml of cisplatin, b1-blank, b2-42.4 uM of gefitinib was used, b3-3.9 ug/ml of cisplatin, b4-1 ug/ml of cisplatin + 10 uM gefitinib, b5-1 ug/ml of cisplatin + 20 uM of gefitinib, b6-1 ug/ml of cisplatin + 30 uM of gefitinib

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 > Results Top

Growth inhibition

Cells of both cell lines were exposed to different concentrations of cisplatin and gefitinib, each alone for 24 h as shown in [Figure 2], dose-response curves were plotted to calculate the IC 50 values, [Table 1] shows IC 50 values, tca8113/CDDP is more resistant to cisplatin than tca8113, and with a slightly more resistance to gefitinib than tca8113.
Figure 2: Dose-inhibition curves show higher resistance of tca8113/CDDP than tca8113 to both cisplatin and gefitinib, when exposed for 24 h

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Table 1: IC50 values for both drugs on both cell lines

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Western blot assay

Six Dishes of each cell line were cultured for 24 h with drugs as shown in [Figure 1]. The expression of Ras, Raf and p-ERK1/2 was detected; all proteins were affected by increasing doses for both cell lines. There is no significant difference when a mix of low concentrations of drugs compared to using each drug alone, so down-regulation of those proteins is in a dose-dependent manner, tca8113/CDDP was more resistant than tca8113, but combinations of low concentrations "lower than the IC 50" was more effective than using each drug alone.

 > Discussion Top

Many studies have shown a relation between EGFR and the development of oral cancer. [8] EGFR and its downstream pathways are targeted for curing cancer. In our study, we have focused on evaluating the effect of EGFR blocking drugs on oral cancer cells. It has been shown that blocking the pathway, which is started by phosphorylation of EGFR led to inhibition of growth, [9],[10] particularly, we targeted the Ras/Raf/MEK pathway because of the strong evidence of the over-expression of these proteins in many cancers. In addition, the expression of mutated Ras has been found in 20-30% of human cancers, [11] as well, Raf and MEK mutations have been reported in many cancers. [11],[12]

Anticancer drugs that inhibit those proteins have shown promising evidence as cancer treatment agents. [4] Ras and Raf are known to be proto-oncogenes. Ras seems to play a significant role in the downstream proteins activated by EGFR. Ras in turn activates Raf, which activates the MAPK/ERK kinase (MEK) 1/2, which then activates ERK1/2. [13],[14]

Cisplatin is one of the commonly used anticancer in the treatment of oral cancer. The mechanism of its action is mainly by regulating cell cycle, damage repair and apoptosis. [15],[16],[17] However, oral cancer cells have been shown to develop resistance to drugs, therefore, halting its effect as an anticancer drug, [6] this explains the difference between IC 50 values of cisplatin for the two cell lines in this study, tca8113/CDDP and tca8113. Tca8113/CDDP cells managed to develop an unclear resistance mechanism to cisplatin. However, some speculations are: Decreased accumulation of cisplatin in the cell, increased levels of glutathione and other intracellular nucleophiles, increased DNA repair and alterations of the apoptotic pathways. [16],[17],[18],[19] Cisplatin has been proven to induce the expression of EGFR; this is explained by the fact that MAPK is involved in the resistance mechanism of cisplatin. [18] Cells create mechanisms of survival employing EGFR; [15] this is obvious in [Figure 1] by the slight over-expression of the EGFR downstream proteins "a3, b3 [Figure 1]."

Gefitinib Iressa, AstraZeneca, UK and Sweden has been approved for clinical usage in many countries, including the united states, Canada, Japan and others, for the treatment of nonsmall cell lung carcinoma, gefitinib is an orally active low molecular weight synthetic anilinoquinazoline, which inhibits many tyrosine kinases, especially the EGFR, [20] gefitinib has proven to be effective in inhibiting EGFR and its downstream pathway (Ras/Raf/MAPK), [20],[21],[22] in our study, we have shown that the (Ras/Raf/MAPK) proteins have been inhibited in response to increasing concentrations of gefitinib, combination of cisplatin with gefitinib proved to be effective in reducing the expression of Ras, Raf and ERK1/2 in both cell lines, but combination have shown more effect on both cell lines when both drugs added at the same time.

 > Acknowledgements Top

This study was kindly supported by The Natural Science Foundation of Heilongjiang Province (Item Number, 2010025).

 > References Top

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Hirsch FR, Varella-Garcia M, Cappuzzo F, McCoy J, Bemis L, Xavier AC, et al. Combination of EGFR gene copy number and protein expression predicts outcome for advanced non-small-cell lung cancer patients treated with gefitinib. Ann Oncol 2007;18:752-60.  Back to cited text no. 9
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McCubrey JA, Steelman LS, Chappell WH, Abrams SL, Montalto G, Cervello M, et al. Mutations and deregulation of Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades which alter therapy response. Oncotarget 2012;3:954-87.  Back to cited text no. 11
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  [Figure 1], [Figure 2]

  [Table 1]


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