Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy
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, 2...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2012
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| Цитувати: | Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy / A.L.S. Galbiatti, H.C. Caldas, J.A. Padovani Junior, E.C. Pavarino, E.M. Goloni-Bertollo // Experimental Oncology. — 2012. — Т. 34, № 4. — С. 367-369. — Бібліогр.: 22 назв. — англ. |
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irk-123456789-1398562018-06-22T03:05:11Z Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy Galbiatti, A.L.S. Caldas, H.C. Padovani Junior, J.A. Pavarino, E.C. Goloni-Bertollo, E.M. Short communications 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. 2012 Article Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy / A.L.S. Galbiatti, H.C. Caldas, J.A. Padovani Junior, E.C. Pavarino, E.M. Goloni-Bertollo // Experimental Oncology. — 2012. — Т. 34, № 4. — С. 367-369. — Бібліогр.: 22 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/139856 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
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English |
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Short communications Short communications |
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Short communications Short communications Galbiatti, A.L.S. Caldas, H.C. Padovani Junior, J.A. Pavarino, E.C. Goloni-Bertollo, E.M. Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy Experimental Oncology |
| description |
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. |
| format |
Article |
| author |
Galbiatti, A.L.S. Caldas, H.C. Padovani Junior, J.A. Pavarino, E.C. Goloni-Bertollo, E.M. |
| author_facet |
Galbiatti, A.L.S. Caldas, H.C. Padovani Junior, J.A. Pavarino, E.C. Goloni-Bertollo, E.M. |
| author_sort |
Galbiatti, A.L.S. |
| title |
Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy |
| title_short |
Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy |
| title_full |
Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy |
| title_fullStr |
Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy |
| title_full_unstemmed |
Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy |
| title_sort |
sensitivity of human laryngeal squamous cell carcinoma hep-2 to metrotexate chemoterapy |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2012 |
| topic_facet |
Short communications |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/139856 |
| citation_txt |
Sensitivity of human laryngeal squamous cell carcinoma Hep-2 to metrotexate chemoterapy / A.L.S. Galbiatti, H.C. Caldas, J.A. Padovani Junior, E.C. Pavarino, E.M. Goloni-Bertollo // Experimental Oncology. — 2012. — Т. 34, № 4. — С. 367-369. — Бібліогр.: 22 назв. — англ. |
| series |
Experimental Oncology |
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| first_indexed |
2025-07-10T09:14:28Z |
| last_indexed |
2025-07-10T09:14:28Z |
| _version_ |
1837250773581299712 |
| fulltext |
Experimental Oncology ��� �������� ���� ��ecem�er���� �������� ���� ��ecem�er� ��ecem�er� ���
SENSITIVITY OF HUMAN LARYNGEAL SQUAMOUS CELL
CARCINOMA HEP-2 TO METROTEXATE CHEMOTERAPY
A.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, Brazil
2Laboratory 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, Brazil
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 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 h
MTX dose Concentration Viable cells, M ± SD
Control group 9,260 ± 50
0.25 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.
ACKNOWLEDGMENTS
Fundação 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.
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