Aim: Methotrexate (MTX) is an antifolate agent that acts inhibiting purine and pyrimidine synthesis. The objective of the study was to evaluate the viability of Hep-2 human laryngeal cancer cells to the treatment with MTX chemotherapy in vitro. Methods: Cultured Hep-2 cells were treated with 0.25, 25.0 and 75 μM MTX for 24 h, and their viability was evaluated with Bcl-2-FITC antibody in flow cytometry. Results: The numbers of viable Hep-2 cells after 24 h treatment with 0.25, 25.0 and 75.0 uM MTX were 85.43%, 22.46% and 8.42%, respectively (p < 0.05). Therefore, MTX possesses a dose-dependent effect on viability of Hep-2 cells in vitro. Conclusion: The highest MTX concentration is associated with highest tumor cell sensitivity of human laryngeal cancer cells of Hep-2 line

Caldas, H.C.

Galbiatti, A.L.S.

Goloni-Bertollo, E.M.

Padovani Junior, J.A.

Pavarino, E.C.

Experimental Oncology

Наукова електронна бібліотека періодичних видань НАН України (Vernadsky National Library of Ukraine)

Experimental Oncology ��� �������� ���� ��ecem�er� ���SENSITIVITY OF HUMAN LARYNGEAL SQUAMOUS CELL CARCINOMA HEP-2 TO METROTEXATE CHEMOTERAPYA.L.S. Galbiatti1,*, H. C. Caldas2, J. A. Padovani Junior3, E.C. Pavarino1, E.M. Goloni-Bertollo1,*1Genetics and Molecular Biology Research Unit, Upgem, FAMERP, São José do Rio Preto Medical School, Brazil2Laboratory of Experimental Immunology and Transplantation, Litex, FAMERP, São José do Rio Preto Medical School, Brazil,3Otorhinolaryngology and Head and Neck Surgery Department of University São José do Rio Preto Medical School, FAMERP, BrazilAim: Methotrexate (MTX) is an antifolate agent that acts inhibiting purine and pyrimidine synthesis. The objective of the study was to evaluate the viability of Hep-2 human laryngeal cancer cells to the treatment with MTX chemotherapy in vitro. Methods: Cultured Hep-2 cells were treated with 0.25, 25.0 and 75 μM MTX for 24 h, and their viability was evaluated with Bcl-2-FITC antibody in flow cytometry. Results: The numbers of viable Hep-2 cells after 24 h treatment with 0.25, 25.0 and 75.0 uM MTX were 85.43%, 22.46% and 8.42%, respectively (p < 0.05). Therefore, MTX possesses a dose-dependent effect on viability of Hep-2 cells in vitro. Conclusion: The highest MTX concentration is associated with highest tumor cell sensitivity of human la-ryngeal cancer cells of Hep-2 line.Key Words: cell line, laryngeal cancer, methotrexate, dose-response relationship, flow cytometry. Head and neck cancer �HNC� includes tumors of pharynx� oral cavity and larynx. The treatment of these tumors may �e surgery� radiotherapy and chemotherapy [���]. Methotrexate ����-diamino�N��-methylpteroyl glutamic acid� �MTX� is an antiprolifera-tive and immunosuppressive chemotherapeutic agent widely used against a �road spectrum of diseases� including HNC [�� 5]. It acts via inhi�ition of the synthe-sis and conversion of folate derivatives responsi�le for providing methyl groups for the nucleotides synthesis and �NA methylation reactions [����]. Although che-motherapy presents good results� tumors may develop resistance to antifolate agents. A num�er of factors are critical for a favora�le clinical outcome for MTX therapy� in particular acute toxicity� side effects� and drug resistance development [5� �����]. The current study was undertaken to evaluate in vitro the human larynx squamous cell carcinoma Hep-� cell line sen-sitivity to the MTX treatment. The cell line was cultured in �ul�ecco Medium ��-MEN ����8 medium� Culti-la��� supplemented with ��% fetal �ovine serum �FBS� Cultila��� � μM glutamine �Cultila��� ��� U/ml of peni-cillin� ��� U/ml of streptomycin� � μM sodium pyruvate �Sigma�Aldrich� and � μM non-essential amino acid �Sigma�Aldrich� in a humidified 5% CO�/�5% air at-mosphere at �� °C. MTX concentrations of �.�5 μM� �5 μM� and �5 μM were calculated according to Pai et al. [��]. Hep-� cells were incu�ated with the men-tioned MTX concentrations for �� h� while the control cells were cultured in MTX-free medium. Cell via�ility was measured �y flow cytometry �FACS cali�er- Bec-ton �ickinson Immunocytometry Systems� San José� USA� with dou�le staining with fluorescein isothiocya-nate �FITC�/Bcl-� according to manufacturer’s manual �Santa Cruz Biotechnology� Inc�. Each experiment was performed in triplicate. The normal distri�ution of the samples was verified with Normality tests �Shapiro — Wilk’s test and Kolmogorov — Smirnov test�. Statistical analysis was performed �y nonparametric methods �ased upon the comparison �etween the groups. The effects for MTX concentrations in the cell via�ility were evaluated independently �y Kruskal-Wallis test �Control group x ���5 uM MTX concentration / Control group x �5 uM MTX concentration / Control group x �5 uM MTX concentration�. For comparison of the varia�les �etween groups exposed with MTX and free-MTX group we used the Mann — Whitney test. The Spearman correlation degree �etween varia�les of interest was calculated �y Spearman test. The results of flow cytometry analysis with FITC/Bcl-� dou�le staining showed that ��.�% of control cells were vital� while the num�ers of via�le Hep-� cells after �� h treatment with �.�5; �5.� and �5.� uM MTX were 85.��; ��.�� and 8.��%� respectively �Figure� Ta�le� �p < �.�5�. The Shapiro — Wilk’s test indicated that there was a normal distri�ution for the groups �p = �.�8��. The Kolmogorov — Smirnov test confirmed that all samples presented significance level of 5% for groups �K-S = �.�8�; p = �.��8� �Ta�le�. The Kruskal-Wallis test indicated a significant effect of MTX in the cell via�ility �H = �.��� p = �.����. The value of Mann — Whitney test showed significant results �p= �.�����. The Spearman correlation �etween frequencies of cells exposed with MTX and unexposed showed an interaction �etween these cells �r = �.5��. Our study confirmed that cells were more sensitive and �ecame less resistant to the MTX chemotherapy as dose was increasing. More-over� there was a correlation �etween cells exposed frequencies with MTX and unexposed cells; literature Received: April 17, 2012. *Correspondence: E-mail: analivia_sg@yahoo.com.br; eny.goloni@famerp.br  Abbreviations used: HNC — head and neck cancer; MTX — metho-trexate.Exp Oncol ������� �� ���������8 Experimental Oncology ��� �������� ���� ��ecem�er�data show MTX concentration correlates with drug therapeutic efficacy [��]. These results suggest that MTX has su�stantial antiproliferative activity and is used effectively as a chemotherapy agent in the treatment for solid-organ neoplasms and the treatment is more efficient when the dose is increased. However� the pi-lot study of Pai et al. [��] that evaluated the sensi�ility of oral cancer cells to MTX in vitro and its association with clinical response to MTX in oral cancer showed that there is differential sensitivity to MTX among the various tumor cells in the in vitro assay� and these data had significant correlation when compared with clinical outcome for � out of �� patients. MTX is an antimeta�o-lite� analogous to folate� that competitively inhi�its di-idrofolatoredutase ��HFR� enzyme activity� essential for nucleotides purines and thymidylic acid �iosynthesis� interfering with �NA synthesis. [��� �8� ��]. Although we found that MTX treatment is highly effective in HNC cells� data confirm that high-dose MTX schemes may arrest normal epidermal cell proliferation and cause direct cell toxicity [��]. Toxicity is increased �y folic acid deficiency or �y medications such as �ar�iturates and nitrofurantoin� which impair folic acid a�sorption [��� ��]. However� it has �een documented that folic acid �� to 5 mg/day� supplementation helps to prevent MTX associated toxicities and concomitant use of ei-ther folic acid with methotrexate has no impact on the therapeutic efficacy of MTX in multiple clinical trials and meta-analyses [��� ��]. Figure. Flow cytometry analysis of Hep-� cells treated with �.�5� �5 and �5 mM MTX �b� c� d respectively� and control cells �a�. The cells in the “R�” �lock are cells non-via�le and cells in the “R�” are via�le. Cell via�ility was evaluated �y dou�le staining with fluorescein isothiocyanate �FITC� la�el Bcl-� ����: sc-5���; Pink cells are via�le� �lue cells — non-via�le Table. Viability of Hep 2 cells treated with MTX for 24 hMTX dose Concentration Viable cells, M ± SDControl group 9,260 ± 500.25 mM 7,910 ± 26*25 mM 2,080 ± 44*75 mM 780 ± 30** The difference is significant compared to the control (p < 0.05).Research a�out chemosensitivity is important to screen new therapeutic agents� identify patterns of sensi�ility for different tumor types� to select chemotherapy regimens to individual patients and improvement in life quality [��]. We conclude that the highest MTX concentration is associated with highest tumor cells sensi�ility; as a consequence� the know-ledge of the drug sensi�ility can do significant impact in decision-making and treatment.ACKNOWLEDGMENTSFundação de Amparo а Pesquisa do Estado de Sгo Paulo �FAPESP� for their financial support �Nє ����/�����-�� ����/�����-��; Prof. �r. Moacir F. Godoy for their help in statistical analysis; Profa. �ra Eloiza Helena Tajara for providing of the cell line; On-cology �epartment� Hospital de Base� São José do Rio Preto for providing of the Methotrexate Chemothera-peutic; CNPQ �National Counsel of Technological and Scientific �evelopment� and FAMERP/FUNFARME.REFERENCES1. Haddad R, Annino D, Tishler RB. Multidisciplinary approach to cancer treatment: focus on head and neck cancer. Dent Clin N Am 2008; 52: 1–17. 2. Marcu LG, Yeoh E. A review of risk factors and genetic alterations in head and neck. Clin Oncol 2009; 135: 1303–14.3. Ignoffo RJ, Rosenbaum EH. What happens in che�motherapy. In: Ko AH, Dollinger M, Rosenbaum EH, eds. Everyone’s Guide to Cancer Therapy, Revised 5th Edition. Kansas City, MS: Andrew McMeel Publishing; 2008. 4. Moura JA, Valduga CJ, Tavares ER, et al. Novel formu�lation of a methotrexate derivative with a lipid nanoemulsion. Int J Nanomedicine 2011; 6: 2285– 95.5.  Am er ican  Can cer  So ciety.  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Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy

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Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy

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