Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate
Aim: The present study was directed on the assessment of the response of treatment-naive oral cancer cells to methotrexate (MTX) in vitro and clinical response to MTX therapy. Methods: A pilot study of in vitro evaluation of MTX response of oral cancer cells from 10 patients was conducted using a...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2009
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| Цитувати: | Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate / R.B. Pai, R.M. Lalitha, S.B. Pai, S.V. Kumaraswamy, N. Lalitha, M.K. Bhargava // Experimental Oncology. — 2009. — Т. 31, № 2. — С. 118–120. — Бібліогр.: 14 назв. — англ. |
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irk-123456789-1357172018-06-16T03:11:02Z Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate Pai, R.B. Lalitha, R.M. Pai, S.B. Kumaraswamy, S.V. Lalitha, N. Bhargava, M.K. Short communications Aim: The present study was directed on the assessment of the response of treatment-naive oral cancer cells to methotrexate (MTX) in vitro and clinical response to MTX therapy. Methods: A pilot study of in vitro evaluation of MTX response of oral cancer cells from 10 patients was conducted using a cell viability assay to determine the sensitivity/resistance to MTX. Quantitative in vitro data were correlated to the clinical outcome to MTX therapy. Results: A positive correlation was observed between the effect of MTX on tumor cells in vitro and clinical response for 7 out of 10 patients. Conclusions: Observations from the proof-of-principle pilot study suggests that oral cancer cells have intrinsically variable response toMTX. Confirmation ofthese findings with a larger cohort of patients could aid in the development of individualized therapies for this class of malignancy. 2009 Article Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate / R.B. Pai, R.M. Lalitha, S.B. Pai, S.V. Kumaraswamy, N. Lalitha, M.K. Bhargava // Experimental Oncology. — 2009. — Т. 31, № 2. — С. 118–120. — Бібліогр.: 14 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/135717 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 |
| language |
English |
| topic |
Short communications Short communications |
| spellingShingle |
Short communications Short communications Pai, R.B. Lalitha, R.M. Pai, S.B. Kumaraswamy, S.V. Lalitha, N. Bhargava, M.K. Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate Experimental Oncology |
| description |
Aim: The present study was directed on the assessment of the response of treatment-naive oral cancer cells to methotrexate (MTX)
in vitro and clinical response to MTX therapy. Methods: A pilot study of in vitro evaluation of MTX response of oral cancer cells
from 10 patients was conducted using a cell viability assay to determine the sensitivity/resistance to MTX. Quantitative in vitro data
were correlated to the clinical outcome to MTX therapy. Results: A positive correlation was observed between the effect of MTX
on tumor cells in vitro and clinical response for 7 out of 10 patients. Conclusions: Observations from the proof-of-principle pilot
study suggests that oral cancer cells have intrinsically variable response toMTX. Confirmation ofthese findings with a larger cohort
of patients could aid in the development of individualized therapies for this class of malignancy. |
| format |
Article |
| author |
Pai, R.B. Lalitha, R.M. Pai, S.B. Kumaraswamy, S.V. Lalitha, N. Bhargava, M.K. |
| author_facet |
Pai, R.B. Lalitha, R.M. Pai, S.B. Kumaraswamy, S.V. Lalitha, N. Bhargava, M.K. |
| author_sort |
Pai, R.B. |
| title |
Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate |
| title_short |
Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate |
| title_full |
Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate |
| title_fullStr |
Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate |
| title_full_unstemmed |
Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate |
| title_sort |
analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2009 |
| topic_facet |
Short communications |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/135717 |
| citation_txt |
Analysis of in vitro and in vivo sensitivity of oral cancer cells to methotrexate / R.B. Pai, R.M. Lalitha, S.B. Pai, S.V. Kumaraswamy, N. Lalitha, M.K. Bhargava // Experimental Oncology. — 2009. — Т. 31, № 2. — С. 118–120. — Бібліогр.: 14 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-09T23:58:20Z |
| last_indexed |
2025-07-09T23:58:20Z |
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1837215779184967680 |
| fulltext |
118 Experimental Oncology 31, 118–120, 2009 (June)
Resistance to anticancer drugs used in the clinic
has led to the persistence of tumor growth and the
failure of chemotherapeutic regimens. Methotrexate
(MTX) has been used as a chemotherapeutic agent
for the management of tumors of varied origins. The
patients presenting with oral carcinoma at our institute
are predominantly habitual chewers of pan, areca
nut and tobacco [1, 2]. Characterization of this class
of neoplasia with unique etiology showed that the ma-
jority of these neoplasia are squamous cell carcinoma
(SCC) and they express certain tumor-specific mar-
kers such as the carcinoembryonic antigen [3]. For oral
cancers patients (prevalently of T3/T4 stages), MTX
is administered to reduce the tumor mass for further
management with radiotherapy and/or surgery and
as a palliative option. Earlier studies on administration
of MTX as a single agent or in combination with other
chemotherapeutic agents as well as other modalities
of therapy such as radiation has shown varying de-
grees of success for head and neck cancers [4–6].
A number of factors are critical for a favorable
clinical outcome of MTX therapy. These include ele-
vated levels of the target, dihydrofolate reductase
(DHFR), diminished polyglutamation and transport
of MTX as well as altered binding affinity of DHFR
to MTX. Translational control of DHFR, including feed
back mechanisms in DHFR biosynthesis could also
contribute to MTX resistance [7, 8]. Multiple mecha-
nisms for MTX resistance in a clinical setting for head
and neck carcinoma has been proposed [9], and the
implications of the various pathways for clinical MTX
resistance has also been reviewed [10].
The current study was undertaken to evaluate the
sensitivity of oral cancer cells to MTX in vitro and its
association with clinical response to MTX in oral cancer
patients.
Patient selection and treatment. Patients were
treated at the Dental Division of Kidwai Memorial Insti-
tute of Oncology, Bangalore, India and were randomly
included in the study. Informed patient consents were
obtained, and approved treatment protocols were admi-
nistered. In addition, patients who had advanced disease
were put through a screening committee to start che-
motherapy for palliation. All the patients (35 to 65 years
old) recruited in the study received chemotherapy with
weekly MTX injections at the dose of 50 mg IM. A maxi-
mum of five injections were administered. During the
treatment period, the patients were monitored for total
WBC count, nausea, vomiting and mucositis. All clinical
assessments of the response were made after the che-
motherapy phase to evaluate the correlation between the
in vitro sensitivity to MTX and tumor response. Some pa-
tients subsequently received radiotherapy, two patients
received radiotherapy prior to MTX therapy.
Criteria for clinical response. The following crite-
ria were used for the assessment of clinical response.
Complete response: total disappearance of the clini-
cally viable lesion; partial response: > 50% reduction
in tumor size; minimal response: < 50% reduction
in tumor size; and no response: stable disease.
Chemicals used in the assay. Trypan blue, Col-
lagenase IV, Hanks Balanced Salt Solution (HBSS)
were all obtained from Sigma Chemical Co (St. Louis,
MO, USA). MTX was a kind gift from Dr. R.M. Lalitha.
MTX dissolved in L-15 tissue culture media was used
in the study. The L-15 tissue culture medium with
composition identical to that of GIBCO BRL, USA was
used. Final assay medium contained L-15 medium
supplemented with 10% fetal bovine serum (FBS).
Preparation of cell suspensions from tumor
tissue. Tumor biopsy samples prior to treatment were
ANALYSIS OF IN VITRO AND IN VIVO SENSITIVITY OF ORAL
CANCER CELLS TO METHOTREXATE
R.B. Pai1, §, R.M. Lalitha1, 2, §, S.B. Pai1, §, *, S.V. Kumaraswamy1, 3, N. Lalitha1, M.K. Bhargava1
1Kidwai Memorial Institute of Oncology, Hosur Road, Bangalore 560029, India
2Department of Oral and Maxillofacial Surgery, M.S. Ramaiah Dental College and Hospital, Bangalore
560054, India
3V.S. Dental College and Hospital, Bangalore 560004, India
Aim: The present study was directed on the assessment of the response of treatment-naive oral cancer cells to methotrexate (MTX)
in vitro and clinical response to MTX therapy. Methods: A pilot study of in vitro evaluation of MTX response of oral cancer cells
from 10 patients was conducted using a cell viability assay to determine the sensitivity/resistance to MTX. Quantitative in vitro data
were correlated to the clinical outcome to MTX therapy. Results: A positive correlation was observed between the effect of MTX
on tumor cells in vitro and clinical response for 7 out of 10 patients. Conclusions: Observations from the proof-of-principle pilot
study suggests that oral cancer cells have intrinsically variable response to MTX. Confirmation of these findings with a larger cohort
of patients could aid in the development of individualized therapies for this class of malignancy.
Key Words: oral cancer, methotrexate, intrinsic, drug sensitivity/resistance.
Received: February 20, 2009.
§Equal contribution.
*Correspondence: Fax: 4048948266
E-mail: balakrishna.pai@ce.gatech.edu
Abbreviations used: 5-FU — fluorouracil; DHFR — dihydrofolate re-
ductase; HBSS — Hanks balanced salt solution; MTX — methotrexa-
te; SCC — squamous cell carcinoma; WBC — white blood cells.
Exp Oncol 2009
31, 2, 118–120
Experimental Oncology 31, 118–120, 2009 (June) 119
placed in HBSS at 4 °C. To generate tumor cell suspen-
sions from the biopsies, the tissue was minced using
scissors in a watch glass. The minced tissue was sus-
pended in 0.075% collagenase for 30 min at 37 °C to ob-
tain single cell suspensions for the assay. The larger
clumps were discarded after the initial centrifugation
(27 x g). The supernatants were further subjected
to a centrifugation at 475 x g. An aliquot of the cell sus-
pension was mixed with equal volume of trypan blue
solution, mixed gently and placed in a hemacytometer,
and the number of viable cells in the suspension.
Drug sensitivity assay. Cultured oral cancer cells
were maintained in L-15 media supplemented with
10% FBS, and treated at three different concentrations
of MTX (0.25 µM, 25 µM, and 75 µM) for 24 h at 37 °C.
The highest concentration was selected based on the
use of similar concentrations for MTX-resistant human
cells in earlier studies [11].
Assessment of cell viability in vitro. After
24 h of incubation with MTX, aliquots of cell samples
were mixed gently with equal volume of trypan blue
dye solution and immediately examined using light
microscopy. Viable cells were counted in triplicates
for each MTX concentration per sample. Results were
presented as mean ± standard deviation.
Criteria for in vitro sensitivity. The cells were
scored as sensitive, moderately sensitive, moder-
ately resistant and resistant, based on the LC50 values
(LC50: lethal concentration inducing 50% of cell death).
If the LC50 was achieved at the lowest concentration
(0.25 µM), the tumor cell populations were considered
as sensitive, whereas inability to attain LC50 even at the
highest concentration used (75 µM) would render them
to be classified as resistant. If LC50 was in the proximi-
ty of 25 µM MTX the tumor cells were designated
as moderately sensitive. Attaining LC50 at the highest
concentration (75 µM) rendered them to be classified
as moderately resistant.
The study was performed on 10 oral tumors
of T4 stage (50%), T3 stage (30%), or T2 stage (20%);
staging from T2N0 to T4N3 (Table 1). Four patients
presented with carcinomas of the buccal mucosa and
four with carcinoma of the alveolus. There was one
presentation with carcinoma of the floor of the mouth,
and one locoregional extension of the carcinoma of the
buccal mucosa to tongue.
Table 1. Clinical characteristics of patient samples used in the study
Patient
No. Age Sex Site of Lesion# Stage
of Tumor Histological type, grade
1 45 F Ca BM T4N3 Squamous carcinoma
2 52 M Ca tongue (L) T2N0 Verrucous
3 42 F Ca BM (R) T3N1 Squamous carcinoma, Gr II
4 35 F Ca alveolus(L) T4N1 Squamous carcinoma, Gr II
5 45 F Ca alveolus(L) T4N1 Squamous carcinoma, Gr III
6 55 F Ca alveolus(L) T3N1 Squamous carcinoma, Gr III/IV
7 58 F Ca alveolus(L) T4N0 Squamous carcinoma, Gr II
8 65 F Ca BM (R) T3N1 Verrucous
9 60 M Ca floor of mouth T4N1 Squamous carcinoma, Gr III
10 35 F Ca BM (R) T2N1 Squamous carcinoma, Gr II
Notes: #BM — buccal mucosa; R — right; L — left.
Differential sensitivity to MTX was observed among
the various tumor cells in the in vitro assay, and these
data were compared to the clinical outcome (Figure
and Table 2). In case 1, tumors cells were found
to be sensitive to MTX, and the laboratory data from
patient 1 corresponded to a good clinical response.
In case 2, in vitro response of tumor cells was modera-
tely sensitive, but clinical response in that patient was
good. In cases 3 and 7, moderate resistance of tumor
cells in vitro and moderate response or good response
in clinical conditions were registered, respectively. Tu-
mor cells from cases 5, 6, 8–10 were resistant to MTX
in vitro, and in all these cases clinical response on MTX
therapy was moderate or absent.
0
20
40
60
80
100
120
140
160
1 2 3 4 5 6 7 8 9 10
Patients
Vi
ab
le
c
el
ls
(%
o
f c
on
tro
l)
0.25 µM
25 µM
75 µM
Figure. The cell viability was assessed by using trypan blue
dye exclusion assay. Mean of triplicate treatments ± standard
deviation is represented. Assays using tumor cells without inclu-
sion of MTX served as controls. Viable cells from controls were
considered as 100% to determine the effect of MTX
Table 2. Correlation between in vitro sensitivity and clinical response
to methotrexate
Patient
No. Treatment Clinical response
to Methotrexate treatment Laboratory analysis (LC50, µM)
1 CT Good response Sensitive (< 0.25)
2 CT Good response Moderate Sensitive ( ~25 )
3 CT Moderate response Moderate resistance ( ~75 )
4 CT 50% response Variable (NE)
5 CT Moderate response Resistant (> 75 )
6 CT Resistant Resistant (> 75 )
7 CT Good response Moderate resistance (< 75 )
8 CT Resistant Resistant (> 75 )
9 RT + CT Resistant Resistant (> 75 )
10 RT + CT Resistant Resistant (> 75 )
Note. NE — not estimated.
Failure of anticancer chemotherapy is largely
attribu ted to intrinsic or acquired resistance to anti-
cancer drugs. Our studies using cells from biopsies
of oral tumors from patients prior to initiation of MTX
treatment depict existence of intrinsic resistance to the
drug in a number of cases.
Our observations with oral carcinoma cells suggest,
for the first time, that the intrinsic resistance to MTX could
be a contributing factor in the lack of response of oral
carcinoma to MTX in the clinic. The implications of various
chemosensitivity assays and their extrapolation to in vivo
scenario have been discussed in detail elsewhere [12, 13].
Studies on drug sensitivity of advanced cancers of gingi-
vo-buccal, tongue and floor of the mouth to va rious drugs
showed that 52% of the tumors tested showed sensitivity
to MTX with a good clinical correlation [14].
Future studies on a larger patient population could
provide valuable information with respect to the genera-
lity of intrinsic MTX resistance in oral cancers in South In-
dia. Further, in vitro–in vivo correlation from such a study
could provide directions for designing individuali zed
therapies for this unique class of neoplasia.
120 Experimental Oncology 31, 118–120, 2009 (June)
ACKNOWLEDGEMENTS
This study was supported by the Indian Council
of Medical Research, New Delhi, India. We thank
Rohith Pai for critical reading of the manuscript and
valuable suggestions.
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