To understand the mechanisms responsible for drug resistance in tongue can-cer, Tca8113 cells derived from moderately differentiated human tongue squamous cell carcinoma were exposed to
Trang 1pingyangmycin-induced drug resistance in human tongue cancer cells
Guopei Zheng1,*, Min Zhou1,*, Xinrong Ou2, Bo Peng1, Yanhui Yu1, Fangren Kong1,
Yongmei Ouyang1and Zhimin He1
1 Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan, China
2 Department of Stomatology, Xiangya Hospital, Central South University, Changsha, Hunan, China
Introduction
Squamous cell carcinoma of the head and neck is the
fifth most common cancer worldwide, and is a
signifi-cant source of cancer morbidity and mortality More
than 500 000 new cases are estimated to occur world-wide every year [1,2] Tongue cancer is the most com-mon type of squamous cell carcinoma of the head and
Keywords
CA9; cDNA microarray; drug resistance;
pingyangmycin; tongue cancer
Correspondence
Zhimin He, Cancer Research Institute,
Xiangya School of Medicine, Central South
University, Xiangya Road #110, 410078
Changsha, Hunan, China
Fax: +86 0731 82355043
Tel: +86 0731 82355041
E-mail: hezhimin2005@yahoo.com
*These authors contributed equally to this
work
(Received 20 June 2010, revised 6 August
2010, accepted 31 August 2010)
doi:10.1111/j.1742-4658.2010.07836.x
Drug resistance is the major obstacle to successful cancer treatment To understand the mechanisms responsible for drug resistance in tongue can-cer, Tca8113 cells derived from moderately differentiated human tongue squamous cell carcinoma were exposed to stepwise escalated concentrations
of pingyangmycin (PYM) to develop the resistant cell line called Tca8113⁄ PYM, which showed over 18.78-fold increased resistance to PYM as compared with Tca8113 cells, and cross-resistance to cisplatin, pirarubicin, paclitaxel, adriamycin, and mitomycin We found that the resistance was not associated with multidrug resistance transporter 1 (p170, p-gp), multi-drug resistance-associated protein 1 and breast cancer resistance protein overexpression, so we hypothesized that Tca8113⁄ PYM cells must have some other resistance mechanism selected by PYM To test this hypothesis, the global gene expression profiles between Tca8113 and Tca8113⁄ PYM cells were compared by cDNA microarray Eighty-nine genes and thirteen expressed sequence tags with differential expression levels between the two cell lines were identified Some differential expression levels were validated with real-time PCR and western blot Furthermore, the functional valida-tion showed that both carbonic anhydrase (CA) inhibitor acetazolamide application and CA9 silencing with CA9 antisense oligonucleotides contrib-ute to the medium pH increase of Tca8113⁄ PYM cells and enhanced PYM chemosensitivity Moreover, both acetazolamide and CA9 antisense oligo-nucleotides significantly increased PYM-induced caspase 3 activation in Tca8113⁄ PYM cells Thus, our study suggests that the resistance of Tca8113⁄ PYM cells is probably associated with CA9 and other differential expression molecules, and that CA9 may be an important marker for pre-diction of PYM responsiveness in tongue cancer chemotherapy
Abbreviations
ADM, adriamycin; ASO, antisense oligonucleotide; Atz, acetazolamide; BCRP, breast cancer resistance protein; BMP2, bone morphogenetic protein 2; CA, carbonic anhydrase; cDDP, cisplatin; DKK1, dickkopf homolog 1; EST, expressed sequence tag; HIF, hypoxia-inducible factor; MDR, multidrug resistance; MDR1, multidrug resistance transporter 1 (p170, p-gp); MMC, mitomycin; MRP1, multidrug resistance-associated protein 1; MT, metallothionein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PARP, poly(ADP-ribose) polymerase; pHe, extracellular pH; pHi, intracellular pH; pNA, p-nitroaniline; PYM, pingyangmycin; VP-16, etoposide; 5-FU, fluorouracil.
Trang 2neck, and the incidence is increasing every year It is
found to be rapidly progressing, to frequently
metasta-size, and to have a poorer prognosis than carcinoma
of other sites in the oral cavity [3] Chemotherapy
plays a very important role in tongue cancer treatment,
especially for patients who are detected at a late stage
or have potential recurrence after surgical procedures
The benefits of chemotherapy include reduction of the
distant metastasis rate, improved survival rate, and
preservation of organ function, whether or not
com-bined with local⁄ regional treatment [4] In the clinic,
pingyangmycin (PYM), cisplatin (cDDP) and
fluoro-uracil (5-FU) are mostly used in chemotherapy of
ton-gue cancer, but the effectiveness of monotherapy with
PYM for preoperative chemotherapy is only about
67% [5,6] Moreover, the therapeutic benefits of
che-motherapy can be attenuated because of intrinsic
and⁄ or acquired drug resistance, especially multidrug
resistance (MDR) ATP-binding cassette transporters
such as MDR transporter 1 (p170, p-gp) (MDR1) have
been reported in some primary tongue squamous cell
carcinomas, and they are chemotherapy-inducible,
showing relevance to drug resistance [7] Accumulating
evidence suggests that multiple complex mechanisms
may be involved, simultaneously or complementarily,
in the emergence and development of drug resistance
in cancers Although some advances in cancer drug
resistance research have been made, indicating that
ATP-binding cassette transporters play important roles
in cancer drug resistance but cannot fully explain the
resistance phenomenon, there are still only a few
stud-ies focusing on tongue cancer
PYM, a water-soluble glycopeptide produced by
Streptomyces pingyangensin, is a new type of cytotoxic
glycopeptide antitumor antibiotic developed in China
in the 1980s It is a member of the bleomycin family,
and is also known as bleomycin A5 It has been found
to reduce the DNA synthesis of cancer cells and cut
off the DNA chain [8] With its wide antitumor
spec-trum and lower toxicity in chemotherapy of malignant
tumors [9], PYM plays a particular curative role in
chemotherapy for treatment of squamous cell
carci-noma, malignant lymphoma, Hodgkin’s disease, and
lymphangioma [10] It is fairly extensively used in
che-motherapy for the treatment of neoplasms in the head
and neck region [10] However, the therapeutic benefits
of PYM can be attenuated in the clinic, because of
intrinsic and⁄ or acquired drug resistance, which is the
major limitation of PYM-based chemotherapy
The mechanism of cellular resistance to PYM is not
fully understood, but it is extremely important to
understand it for successful treatment of tongue
carci-noma In this study, we established a cellular model,
Tca8113⁄ PYM cells, with acquired resistance induced
by PYM, expecting to reveal new molecules related to PYM resistance, and to provide candidate biomarkers
to predict the clinical response to PYM-based chemo-therapy in tongue cancers
Results
Biological characteristics of Tca8113⁄ PYM cells
In order to explore the mechanism responsible for PYM resistance in tongue cancer, in the first step of the pres-ent study we established a PYM-resistant cell line, Tca8113⁄ PYM The Tca8113 ⁄ PYM cell line was obtained by stepwise selection from its sensitive parent cell line with PYM over a period of 2 years At the beginning of induction, cell growth was strongly sup-pressed However, at the end of induction, Tca8113⁄ PYM cells exhibited a stable growth pattern in medium with 0.2 mgÆL)1 PYM After further maintenance for
5 weeks in PYM-free medium, the mean population doubling time was found to be 35.76 ± 4.62 h for Tca8113⁄ PYM cells, as compared with 35.12 ± 4.18 h for Tca8113 cells (no statistically significant difference;
P> 0.05) (Fig 1A) After cells had been treated with different concentrations of PYM from 0 to 800 mgÆL)1 for 48 h, dose–response curves were plotted, as shown in Fig 1B The IC50 values for PYM treatment in Tca8113 and Tca8113⁄ PYM cells were 27.16 ± 1.78 mgÆL)1 and 509.47 ± 37.71 mgÆL)1, respectively (P < 0.01) The resistance index was 18.78, indicating that the PYM-resistant cell line was successfully established The antiapoptotic activity of Tca8113⁄ PYM cells was also measured with Hoechst33258 stain (Fig 1C) Twenty-four hours after treatment with 300 mgÆL)1 PYM, despite increased numbers of apoptotic cells, typically identified as those cells that possess signifi-cantly smaller, condensed and fragmented nuclei under
a fluorescence microscope, in both cell lines, the rate
of apoptosis in Tca8113 cells was much higher than that in Tca8113⁄ PYM cells: 88.10± 7.96% and 15.86± 2.75%, respectively (P < 0.01)
In addition, in order to determine whether the resistance was associated with the overexpression of well-documented resistance-related molecules MDR1, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), RT-PCR was performed (Fig 1D) There was no significant differ-ence in MDR1 or BCRP mRNA level between Tca8113 and Tca8113⁄ PYM cells, and there was no detectable expression of MRP1, indicating that resis-tance to PYM may be associated with some other mole-cules
Trang 3Cross-resistance profiles of Tca8113⁄ PYM cells The sensitivities of both cell lines to cDDP, piraru-bicin, paclitaxel, mitomycin (MMC), adriamycin (ADM), etoposide (VP-16) and 5-FU were determined
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, and the IC50 for each agent was calculated Both cell lines were treated with differ-ent concdiffer-entrations of each agdiffer-ent, and the IC50 values are summarized in Table 1 The results revealed Tca8113⁄ PYM cells showed resistance to cDDP, pira-rubicin, paclitaxel, ADM and MMC, but not to VP-16
or 5-FU, indicating that Tca8113⁄ PYM was a typical MDR model, and that studies on the mechanism of resistance in this cell line have potential significance
Differential gene expression profiles between Tca8113⁄ PYM and Tca8113 cells
Because there were no differences in expression of the well-known resistance-related genes MDR1, MRP1 or BCRP, to identify genes generally involved in PYM
A
B
C
D
4 )
Concentration of PYM (mg·L –1 )
Days
Fig 1 Biological characteristics of Tca8113 ⁄ PYM cells (A) Growth curves for both cell lines were obtained in three independent exper-iments, and showed no difference in the mean doubling time (B) Responses of Tca8113 and Tca8113 ⁄ PYM cells to PYM Tca8113 ⁄ PYM cells were more resistant to the antiproliferative activity of PYM Each point represents the mean of six independent experi-ments (C) The effects of PYM-induced apoptosis on both cell lines were observed with Hoechst33258 stain The results represent three independent experiments, and show that Tca8113 ⁄ PYM cells are much more resistant to PYM-induced apoptosis (D) Relative mRNA expression levels of MDR1, MRP1 and BCRP between both cell lines after normalization to b-actin mRNA levels were deter-mined by RT-PCR RT-PCR analysis was repeated three times, and showed no significant expression difference between the two cell lines for MDR1 or BCRP, and no detectable expression of MRP1.
Table 1 IC50 values (mgÆL)1) for selected agents (mean ± stan-dard deviation, n = 3) The results show MDR characteristic of Tca8113 ⁄ PYM cells RI, resistance index, representing IC50 Tca8113 ⁄ PYM ⁄ IC50 Tca8113
**P < 0.01 and *P < 0.05 versus Tca8113.
Trang 4resistance we compared gene expression profiles
between Tca8113⁄ PYM and Tca8113 cells by cDNA
microarray We excluded genes whose expression was
increased or decreased by less than two-fold in
PYM-resistant cells (as compared with the parent cells) A
total of 89 genes were selected, among which 41 genes
were upregulated (Table 2) and 48 genes were
down-regulated (Table 3) in the Tca8113⁄ PYM cell line In
addition, 13 expressed sequence tags (ESTs) were also
selected, four of which were upregulated and nine of
which were downregulated (Table 4) Interestingly, in our microarray data, there were also no significant dif-ferences in expression of MDR1, MRP1 or BCRP between these two cell lines, in agreement with the results of RT-PCR, so we considered that the resis-tance may be related to a number of other differential genes associated with a variety of cellular functions, such as those encoding carbonic anhydrase (CA) 9, metallothionein (MT) 2A, bone morphogenetic pro-tein 2 (BMP2), and dickkopf homolog 1 (DKK1)
Table 2 Genes with upregulated expression in Tca8113 ⁄ PYM cells.
11 PPP2R2B NM_004576 2.618 b-Isoform of regulatory subunit B55, protein phosphatase 2 isoform a
growth factor receptor substrate 2)
tract-binding protein associated)
4-hydroxylase), a-polypeptide I
Trang 5Confirmation of differential expression by
real-time PCR and western blot
To confirm the results of the microarray, we carried
out quantitative real-time PCR and western blot, each
been repeated three times Eight differential expression genes and ESTs were examined at the mRNA level (Fig 2A) A good correlation between the real-time PCR results and the microarray data was seen For example, the mean fold changes in upregulation as
Table 3 Genes with downregulated expression in Tca8113 ⁄ PYM cells.
N-acetylgalactosaminyltransferase 10
Trang 6determined by microarray analysis and by real-time
PCR were, respectively, 4.993 and 5.584 for CA9,
4.933 and 4.291 for BMP2, 3.772 and 4.815 for
CD237904, and 2.544 and 2.884 for AL707095 The mean fold changes in downregulation as determined by microarray analysis and by real-time PCR were, respectively, 2.320 and 2.794 for DKK1, and 2.415 and 2.895 for BC037851 Also, the expression of MT2A and CA9 was further tested by western blot (Fig 2B), exploring their functional linkages to biochemical mechanisms that could be related to PYM resistance Both real-time PCR and western blot analyses con-firmed the microarray results
CA9 interference sensitizes Tca8113⁄ PYM cells to PYM
Recent studies have demonstrated that CA9 over-expression represents biological tumor aggressiveness, and is associated with poor clinical outcome in several tumors, including head and neck, cervix, kidney and lung cancers However, the nature and mechanism of CA9 involvement are not well established; in particu-lar, direct evidence in drug resistance is lacking In our present study, CA9 expression was upregulated in Tca8113⁄ PYM cells, and we conducted two series of experiments to investigate its role in PYM resistance
In the first series of experiments, the CA function inhibitor acetazolamide (Atz) was used First, we determined 800 lm as the concentration of Atz to be used for the follow-up experiments (data not shown) Then we measured the pH of the culture medium affected by Atz The pH of Tca8113⁄ PYM cells, 6.37 ± 0.11, is much lower than that of Tca8113 cells, 6.65 ± 0.16, indicating that CA9 does actually play a role After Atz administration, the medium pH of Tca8113⁄ PYM cells was significantly increased, by about 0.36 units (Fig 3A) We found that 800 lm Atz enhanced the sensitivity of Tca8113⁄ PYM cells to PYM, with an IC50 reduction from 509.47 ± 37.71 mgÆL)1 to 89.41 ± 9.33 mgÆL)1 (P < 0.01), but had no effect on their parent cell line, Tca8113 (Fig 3B) In addition, we observed the effect of Atz
on PYM-induced apoptosis with Hoechst33258 stain, and found that Atz combined with 100 mgÆL)1 PYM increased the proportion of apoptotic Tca8113⁄ PYM cells by about 56% (Fig 3C) The observation of apoptosis was validated by the detection of the func-tional states of caspase 3 and poly(ADP-ribose) poly-merase (PARP) at the protein level 24 h after the respective treatments, suggesting that Atz can signifi-cantly enhance 100 mgÆL)1 PYM-induced caspase 3 and PARP cleavage (Fig 3D)
To further validate the functional role of CA9 upregulation in Tca8113⁄ PYM cells, in the second ser-ies of experiments inhibition of CA9 gene expression
Table 4 ESTs with downregulated ⁄ upregulated expression in
Tca8113 ⁄ PYM cells.
UniGene Cluster ID
Fold downregulation⁄
upregulation
A
B
Fig 2 Validation of microarray results (A) Real-time PCR: relative
expression levels of selected transcripts are shown in a fold scale
between Tca8113 ⁄ PYM and Tca8113 by normalizing against
b-actin Bars and standard errors representing relative expression
of tested genes normalized against b-actin in the data were
obtained from three independent experiments (B) Using western
blot, we validated the protein expression levels of MT2A and CA9,
using a-tubulin as a loading control The figure represents three
independent experiments.
Trang 7with CA9 antisense oligonucleotides (ASOs) was
employed Tca8113⁄ PYM cells were transfected with
CA9 ASOs, and western blot analysis showed that
ASO 2# markedly downregulated CA9 expression, so
the ASO 2# was selected for further study (Fig 4A)
CA9 ASO transfection elevated the medium pH of
Tca8113⁄ PYM cells by 0.3 units (P < 0.01)
(Fig 4B) and enhanced PYM chemosensitivity, with a
significant decrease in the IC50 value from 509.47 ±
37.71 mgÆL)1 to 94.78 ± 9.62 mgÆL)1 (P < 0.01)
(Fig 4C), and an increase of 52% in the proportion
of apoptotic cells induced by 100 mgÆL)1 PYM
(Fig 4D); the control scrambled ASO had no
signifi-cant effects on the induction of antiproliferation or apoptosis by 100 mgÆL)1 PYM Caspase 3 and PARP cleavage was also detected (Fig 4E), suggesting that silencing of CA9 expression could suppress the antia-poptotic activity of Tca8113⁄ PYM cells to enhance the PYM effect
To further observe the enhanced effect of CA9 inter-ference on PYM-induced Tca8113⁄ PYM apoptosis, cas-pase 3 activity was investigated Tca8113⁄ PYM cells were pretransfected with scrambled ASO and CA9 ASO, and then incubated in 100 mgÆL)1PYM Simulta-neously, Tca8113⁄ PYM cells were treated with
100 mgÆL)1 PYM alone or combined with Atz After
C
D
Fig 3 The effect of inhibition of CA9 function by Atz on PYM activity Every experiment was repeated three times, and bars and standard errors in the data were obtained from three independent experiments (A) The pH change of Tca8113 and Tca8113 ⁄ PYM cells with or with-out Atz treatment The pH of the culture medium for Tca8113 ⁄ PYM cells was much lower than that for Tca8113 cells Atz clearly increased the pH of culture medium for Tca8113 ⁄ PYM cells, but not for Tca8113 cells 4, versus Tca8113, P < 0.05; q, versus Tca8113, P > 0.05; h, versus Tca8113 ⁄ PYM, P < 0.01 (B) The dose–inhibition rate curve plotted from MTT assay results Atz significantly enhanced the effect of PYM on Tca8113 ⁄ PYM cells, with a marked reduction in IC50 value, but not on Tca8113 cells (C) Atz significantly enhanced PYM-induced apoptosis of Tca8113 ⁄ PYM cells as shown by Hoechst33258 stain, and indicated with arrows, representing three independent experiments (D) Caspase 3 and PARP cleavage represent molecular effects of Atz combined with PYM on Tca8113 ⁄ PYM cells.
Trang 824 h of treatment, caspase 3 activity was determined As
shown in Fig 4F, Atz and CA9 ASO significantly
increased PYM-induced caspase 3 activity in Tca8113⁄
-PYM cells (P < 0.01) as compared with untreated cells
Discussion
A major problem in the clinical chemotherapeutic
treatment of cancer is intrinsic or acquired resistance
to current chemotherapeutic agents [11], particularly
the acquisition of MDR This underlines the critical
importance of exploring the molecular mechanisms
involved in the drug resistance of cancer cells for
improving current treatments in the clinic
PYM is widely used in the treatment of various squamous cell tumors, including tongue cancer This stresses the need to elucidate the mechanism of drug resistance induced by PYM Here, we established an isogenic PYM-resistant variant, Tca8113⁄ PYM, from the tongue cancer cell line Tca8113 to compare their gene expression profiles directly cDNA microarray analysis, which is a powerful technology for the identi-fication of well-documented and novel genes associated with response or resistance to chemotherapeutic agents, was used [12], and revealed that 41 genes were upregulated and 48 genes were downregulated in the Tca8113⁄ PYM cell line However, there were no differ-ences in the expression of MDR1, MRP1 or BCRP
A
B
D
E
F
C
Fig 4 The effect of CA9 ASO on PYM.
Every experiment was repeated three
times, and bars and standard errors in the
data were obtained from three independent
experiments (A) The CA9 expression
change mediated by ASO showed that CA9
ASO2# significantly reduced the CA9 protein
level So the CA9 ASO 2# was selected for
the follow-up experiments (B) CA9 ASO
increased the medium pH of Tca8113 ⁄ PYM
cells, but scrambled ASO did not *:versus
Tca8113 ⁄ PYM, P < 0.01 (C, D) CA9 ASO
significantly decreased cell viability, with a
marked reduction in IC50 value, and
increased the apoptotic activity induced by
PYM on Tca8113 ⁄ PYM cells (E) Western
blot analysis revealed that CA9 ASO could
enhance PYM-induced caspase 3 activation
and subsequent PARP cleavage (F) Effect
of CA9 ASO on PYM-induced caspase 3
activation on Tca8113 ⁄ PYM cells The
rela-tive activation of caspase 3 shown was
cal-culated from the average of three
experiments Each value is expressed as
ratio of caspase 3 activation level to
untreated level, and the untreated level
was set to 1 *versus untreated, P > 0.05;
**versus untreated, P < 0.01.
Trang 9between the two cell lines, revealing that the drug
resis-tance of tongue cancer induced by PYM may be
related to some other molecules, such as CA9
CA9 is a zinc metalloenzyme catalyzing the
revers-ible conversion of CO2to bicarbonate and a proton, is
a cell surface glycoprotein, and reduces local
extracel-lular pH [13] CA9 overexpression has been identified
in a number of solid tumors, including renal
carcino-mas and, particularly, clear cell adenocarcinocarcino-mas,
cer-vical squamous carcinomas, ovarian carcinomas,
colorectal carcinomas, esophageal carcinomas, bladder
carcinomas and non-small cell lung carcinomas CA9
is strongly induced by hypoxia via the transcription
factor hypoxia-inducible factor (HIF)-1 or by an
HIF-independent pathway, such as the activation of
oncog-enes (Ras, SRC, and PI3K) or inactivation of tumor
suppressor genes (PTEN and p53), and is thought to
play a role in the regulation of cancer cell
prolifera-tion, cell transformation and survival under normoxia
or hypoxia, making it a potential target for cancer
therapy [14–16] However, the array analysis showed
no significant differences in expression of the above
genes between Tca8113 and Tca8113⁄ PYM cells, and
the western blot analysis of HIF-1a also showed no
difference (data not shown) These negative data imply
that a different mechanism, such as methylation or
microRNA, is responsible for the upregulation of CA9
expression Michael et al suggested that CA9
expres-sion in squamous cell head and neck tumor had a
significant relationship with resistance to
chemoradio-therapy [17], in support of our study, but there was a
lack of direct evidence for the mechanism In our
pres-ent study, CA9 expression was much higher in
Tca8113⁄ PYM cells and the extracellular pH was
much lower in the same incubation conditions
Gener-ally, solid tumors maintain a high intracellular pH
(pHi) but a low extracellular pH (pHe) Adaptation of
tumor cells to hypoxia and acidosis is a critical driving
force in tumor progression and metastasis [18,19]
Tumor cells have developed key strategies to regulate
their pHi, because a pHi variation of 0.1 can disrupt
multiple biological functions, including ATP
produc-tion, protein synthesis, cell proliferaproduc-tion, migraproduc-tion,
and apoptosis [20–22] Whether the adaptation
corre-lates with the drug resistance needs further
investiga-tion Here, both CA9 function inhibition and CA9
expression silencing elevated the pHe of Tca8113⁄
PYM cells, suggesting that CA9 function really
con-tributed to the regulation of pHe Moreover, CA9
interference significantly decreased the IC50 of PYM
in Tca8113⁄ PYM cells, and enhanced the effect of
PYM-induced cell apoptosis and caspase 3 activity
However, the exact mechanism is still unclear Chiche
et al [23] showed that forced expression of CA9 con-tributed to extracellular acidification and to the main-tenance of a more alkaline resting pHi Importantly, the efficiency of caspase activation by cytC was found
to be pH-sensitive, and lower pH contributed to more caspase activation [24] The change in cytosolic pH may play a very important role in regulating the apop-totic process, but whether CA9-mediated drug resis-tance is associated with the maintenance of cytosolic
pH, what happens inside cells after Atz or CA9 ASO
is administered in combination with PYM, and how the overexpression of CA9 occurrs during PYM induc-tion will be investigated in further studies
CA9 plays a very important role in PYM-induced drug resistance, but CA9 interference cannot com-pletely reverse resistance Most of the other genes with altered expression are correlated with tumorigenesis, and some reports have also suggested a role for them
in the drug response For example, MTs, which are known to participate in fundamental cellular processes such as cell proliferation and apoptosis [25,26], have been suggested to protect against toxic and carcino-genic events mediated by a broad range of nonmetal toxicants [27] Several lines of evidence suggest that MTs are chemotherapy inducible [28], and their expression constitutes a protective mechanism that pre-vents the apoptosis induced by cisplatin and doxorubi-cin [29,30] In addition, irinotecan-induced changes in
MT expression correlated with clinical response in gas-tric cancer patients, and MT overexpression modestly increased the resistance of AGS cells to irinotecan [31]
In our previous study, we established a human MT2A recombinant with soluble high-yield expression, and demonstrated its hydroxyl radical-scavenging ability and significant protective role against DNA damage caused by UVC radiation [32] In the present study, the expression of MT2A was upregulated in Tca8113⁄ PYM cells MT2A, as the main isoform of MTs, may closely mediate the resistance of Tca8113⁄ PYM cells to PYM
BMP2 is a member of the transforming growth fac-tor superfamily, and is now recognized as a multipur-pose cytokine that stimulates migration and induces the proliferation and differentiation of many different cell types BMP2 is expressed in a variety of carcinoma cell lines, especially tumors originating from the head and neck [33] Recombinant BMP2 caused an 50% increase in early tumor growth of A549 cells in athy-mic nude athy-mice, whereas BMP2 antagonists inhibited tumor growth by 50% [34] Elaine et al found that BMP2 greatly enhanced blood vessel formation in tumors formed by A549 cells in nude mice, and that,
in vitroin endothelial cells, BMP2 stimulated Smad1⁄ 5,
Trang 10Erk1⁄ 2, and Id1 expression, which was associated with
an increase in tube formation and proliferation, and
suggested that BMP2 could promote tumor growth by
stimulating angiogenesis [35] BMP2 is closely related
to cancer, but its role in chemotherapy has not been
reported Our data showed that expression of BMP2
in Tca8113⁄ PYM cells is 4.933-fold higher than in
Tca8113 cells, so further investigations should be
per-formed
It is well known that the Wnt–b-catenin pathway is
aberrantly activated in many carcinomas [36] As one
of the natural Wnt antagonists, DKK1 simultaneously
binds to LRP5⁄ 6 and the transmembrane proteins
Kre-men 1⁄ 2, and induces LRP endocytosis, which prevents
the formation of Wnt–Frizzled–LRP5⁄ 6 receptor
com-plexes and blocks Wnt–b-catenin signaling [37,38]
DKK1 seems to have antitumor effects independently
of the antagonism of b-catenin–TCF transcriptional
activity in H28 and MS-1 mesothelioma and HeLa
cer-vical cancer cells [39,40] Some studies have
demon-strated that DKK1 is downregulated in colon cancer
[41] and medulloblastoma cells, perhaps because of the
methylation of the promoter, and restoration of DKK1
expression can induce apoptosis and suppress colony
formation [42] As a suppressor of cancer, the
downreg-ulated expression of DKK1 is associated with
chemore-sistance, consistent with previous studies However,
whether its downregulation is the result of methylation
of the promoter in Tca8113⁄ PYM cells and its precise
effects in suppressing cancer or reversing of PYM
resis-tance will be investigated in future studies
In addition, among the ESTs, we have identified a
novel gene termed TCRP1 (tongue cancer drug
resis-tance associated protein; Genebank:EF363480) that
particularly mediates cDDP resistance, and a related
study is being performed (data not shown)
In conclusion, the Tca8113⁄ PYM and Tca8113 cell
lines are useful models for identifying candidate targets
responsible for the mechanism of PYM-induced drug
resistance in tongue cancer Using cDNA microarray
technology, we have identified 89 genes and 13 ESTs
that may be related to PYM-inducible resistance In
particular, CA9 seems to be a potential biomarker,
and its interference may be promising in drug
resis-tance reversion
Experimental procedures
Establishment of the Tca8113⁄ PYM cell line
Tca8113 cells obtained from the China Center for Type
Cul-ture Collection (Wuhan, China) were culCul-tured in RPMI-1640
(Gibco, Carlsbad, CA, USA) containing 10% fetal bovine
was established by intermittent stepwise selection in vitro with PYM (Harbin Bolai Pharmaceutical, Harbin, China)
Tca8113 cells under treatment, the cultures were maintained
by regular changes of medium, with intermittent increases in the PYM concentration until the surviving cells recovered a
were maintained in PYM-free medium for at least 2 weeks
To investigate the cell growth curve, cells were seeded in a
med-ium was replaced with fresh medmed-ium without PYM Four wells were trypsinized each time after 1, 2, 3, 4, 5 and 6 days
of incubation, and the cell number was determined The average cell count obtained at each time point was plotted against the time, and the doubling time was calculated for the exponential growth phase
MTT assay
per well (200 lL per well) for 24 h before use The culture medium was replaced with fresh medium containing antican-cer drug for 48 h Water-soluble MTT (Sigma-Aldrich,
St Louis, MO, USA) was added (20 lL) After 4 h of incuba-tion, the supernatant was discarded and the purple crystals were resuspended in 200 lL of dimethylsulfoxide The absor-bance of each well was read at 570 nm on an ELISA XL (BIOHIT, BP800, Helsinki, Finland) The growth rate was cal-culated as the ratio of the absorbance of the experimental well to that of a blank well, and the IC50 was also calculated
Hoechst stain
Cells in exponential growth were cultured with fresh med-ium in a six-well plate in which the coverslips had been placed After incubation for 24 h, cells were treated with or without agent for 48 h Then, Hoechst33258 was used to detect apoptosis according to a standard procedure, a fluo-rescence microscope was used to observe apoptotic cells, which were typically identified as cells possessing signifi-cantly smaller, condensed and fragmented nuclei, the apop-totic cell number was determined under at least three views for every treated group, and the rate of apoptosis was cal-culated The experiments were repeated three times
RT-PCR and real-time PCR
Total RNA was extracted with a Trizol protocol, and cDNAs from the mRNAs were synthesized with the Super-Script first-strand synthesis system (Fermentas Life Science,