Testicular germ cell tumours (TGCTs) are highly sensitive to cisplatin-based combination
chemotherapy, and most patients with this disease can be cured today. Nevertheless,
10–15% of patients with metastatic disease will not achieve a disease-free survival
with currently available treatment strategies, and finally die of their disease. The
reasons for intrinsic or subsequently developed treatment resistance in these patients
have not yet been fully explored (Mayer et al, 2003). A reduced intratumoral oxygen
tension (hypoxia) has been reported in a variety of malignant tumours (Vaupel and
Hoeckel, 1998; Semenza, 2000), and may limit the effectiveness of cytotoxic drugs.
A positive correlation between the intracellular oxygen tension (pO2) and the efficacy
of a radiotherapy has been described as early as 1931 (Mottram, 1931). These experimental
data are endorsed by more recent clinical findings in patients with cancer of the
uterine cervix, and head and neck tumours undergoing radiotherapy (Hoeckel et al,
1996; Nordsmark et al, 1996). The intratumoral oxygen tension depends at least partly
on the haemoglobin level of the blood (Vaupel and Hoeckel, 1998; Becker et al, 2000).
Under hypoxic conditions, proteins like the vascular endothelial growth factor (VEGF)
and the hypoxia-inducible factor 1 alpha (HIF-1α are upregulated in cancer cells (Zhong
et al, 1999; Kondo et al, 2000; Cooke et al, 2001; López-Barneo et al, 2001). Induction
of antiapoptotic proteins like Bcl-2 or of the multidrug resistance gene (MDR1) product
P-glycoprotein is associated with HIF-1α overexpression, and may lead to resistance
against chemotherapeutic agents (Goldstein, 1996; Zhong et al, 1999; Kinoshita et
al, 2001; Comerford et al, 2002). The loss of apoptotic mechanisms (deprivation of
p53) and loss of DNA mismatch repair (MMR) in hypoxia render cells both hypersensitive
to acquire microsatellite instability and to the development of drug resistance (Lin
et al, 2000, 2001; Kondo et al, 2001).
Most cytotoxic agents show a positive relation between oxygen tension and efficacy
in cell culture. For mitomycin C, higher efficacy in hypoxia has been reported (Kennedy
et al, 1983; Luk et al, 1990; Yamagata et al, 1992; Sanna and Rofstad, 1994). For
cisplatin, the most active drug for TGCTs, the results reported are contradictory,
but seem to indicate an enhanced drug activity under hypoxic conditions in various
models (Liang, 1996; Skov et al, 1998; Kovacs et al, 1999). Other cytotoxic drugs
with described clinical activity in refractory germ cell tumours, such as paclitaxel,
gemcitabine and oxaliplatin, have not yet been tested for their activity under hypoxia
in vitro. In addition, both the choice of the specific chemotherapeutic agent as well
as the tumour type may influence the relative impact of hypoxia in the treatment setting.
Erythropoietin (epoietin) offers the chance to effectively ameliorate anaemia in cancer
patients receiving chemotherapy (Cella et al, 2003). Next to a proven benefit regarding
quality of life, epoietin might potentially affect the efficacy of the anticancer
treatment used by raising the pO2 in tumour tissues. In order to provide the preclinical
rationale for a clinical study with chemotherapy and epoietin in patients suffering
from GCTs, we have investigated the in vitro efficiency of cytotoxic agents with different
modes of action such as alkylating agents (eg, ifosfamide), platin derivatives, antibiotics
(mitomycin C, bleomycin), gemcitabine, topoisomerase I (irinotecan) and II (etoposide)
inhibitors, and the taxane derivative paclitaxel under normoxic and hypoxic conditions
in established TGCT cell lines. Furthermore, the model system chosen here is used
to discuss the relative efficacy of different cytotoxic agents in relation to the
most effective drug cisplatin.
MATERIALS AND METHODS
Anticancer drugs
The drugs used were: cisplatin (CDDP; Bristol-Myers Squibb, München, Germany), oxaliplatin
(Sanofi-Synthelabo GmbH, Berlin, Germany), carboplatin (Bristol-Myers Squibb), gemcitabine
(Lilly Deutschland, Bad Homburg, Germany), etopophos (etoposide phosphate, VP-16;
Bristol-Myers Squibb), bleomycin (Mack, Illertissen, Germany), mitomycin C (medac,
Wedel, Germany), irinotecan (Aventis Pharma, Frankfurt/M., Germany), and 4-hydroperoxyifosfamide
(4-OOH-ifosfamide; Asta Medica, Frankfurt/M., Germany). These agents were dissolved
in distilled water. The semisynthetic agent paclitaxel (Sigma, Deisenhofen, Germany)
from Taxus baccata was dissolved in DMSO (Sigma) and used without exceeding a final
concentration of DMSO 0.1% (v/v), which by itself is not a toxic concentration for
the cell lines studied.
TGCT cell lines and culture conditions
Three established TGCT cell lines derived from human embryonal carcinomas were tested
for their sensitivity towards different chemotherapeutic agents. The TGCT cell line
NTera-2 (NT2/D1, a cell line known to be able to differentiate into neurons); ATCC
CRL-1973 used in this study was maintained in DMEM with 4.5 g l−1 glucose and stable
glutamine (Invitrogen, Karlsruhe, Germany), the 2102 EP cell line (Wang et al, 1981)
and NCCIT (ATCC CRL-2073) were cultured in DMEM/F-12 with 2 mM L-glutamine (Biochrom).
All cell lines were grown with the addition of 10% fetal calf serum (FCS; Biochrom,
Berlin, Germany) and 1% penicillin/streptomycin (Biochrom) at 37°C in a humid atmosphere
containing 5% CO2 as monolayers in 75 cm2 cell culture flasks.
Cell proliferation in normoxia vs hypoxia
For the assessment of doubling times, the cells were cultured in normoxic (20% O2)
and hypoxic (continuous flow of 0.1 l min−1 of a mixture of 94% N2, 5% CO2, and 1%
O2) conditions. Briefly, individual cells were spread out in six-well plates and viable
cells were counted in their logarithmic growth phase after 48 and 70 h to calculate
the population-doubling times under both conditions by trypane blue (0.4%; Sigma)
exclusion.
For determination of cell cycle progression, NT2 and NCCIT cells were grown in 25 cm2
culture flasks in normoxia and hypoxia for 48 h. Further processing was performed
according to the method of Nicoletti et al (1991). In brief, the supernatant and adherent
cells were harvested, washed, and suspended in 0.5 ml hypotonic lysis buffer (0.1%
sodium citrate, 0.1% Triton X-100) containing 25 μl of a 1 mg ml−1 propidium iodide
(PI) stock solution (50 μg ml−1 final concentration). Analysis of the cell cycle phase
was performed by flow cytometry on a FACScalibur (Becton Dickinson, Heidelberg, Germany),
using the CellQuest analysis software.
Determination of pH value of the medium for untreated cells
NT2 and NCCIT cells were cultured with 10 ml complete medium in 25 cm2 cell culture
flasks under normoxic and hypoxic conditions. After 72 h, the pH of the cell medium
was measured using the pH meter model pH330 (WTW, Weilheim, Germany) and compared
with 37°C annealed normoxic and hypoxic medium without cells.
In vitro drug-sensitivity assay
The MTT assay was performed as previously described (Sieuwerts et al, 1995). In brief,
the cell lines NT2, 2102 EP, and NCCIT were rinsed with phosphate-buffered saline
(PBS, Biochrom), trypsinised and resuspended in 1 ml of the appropriate culture medium,
to count the cells in a haemacytometer chamber. In all, 4 × 103 cells/well were seeded
in 96-well plates to ensure their logarithmic growth. Cells were allowed to adhere
over night, serial dilutions of the chemotherapeutic agents were added to octuplicate
wells at concentrations from 1 nM to 0.1 mM. The cells were exposed to the drugs for
additional 72 h under normoxic and hypoxic conditions. Additionally, NT2 and NCCIT
cells were treated with mitomycin C for 72 h under hypoxic conditions using culture
medium adjusted to pH 6.5.
After this, the drug-containing medium was removed and 0.2 ml MTT solution (final
concentration: 0.5 mg/mL MTT; Sigma) was added in ther medium. The plates were incubated
for 2 h and then the medium was removed, 0.1 ml DMSO was added, the plates agitated
for 15 min and the optical density read using a photometer (MRX Revelation, Dynex
Technologies, VWR International, Bruchsal, Germany) at 570 nm.
All experiments were replicated separately twice or more if the values of increase
in IC50 in hypoxia compared to normoxia were greater than 20%, to ensure reproducibility.
The results are expressed as drug concentrations that inhibit cell growth by 50% (inhibitory
concentration; IC50). The IC50 of the tested agents under both conditions were estimated
graphically from the dose–response curves and compared. The relative increase in IC50
in normoxia vs hypoxia was assessed.
Induction and quantification of apoptotic cells
In all, 1 × 105 cells/well for normoxia and 2 × 105–4 × 105 cells/well for hypoxia
were seeded in six-well plates. After overnight preincubation, serial dilutions of
cisplatin and paclitaxel were added to the medium in chosen concentrations for NT2
and 2102 EP cells. Annexin-V labelling of the cells was performed as recommended by
the manufacturer (Roche Diagnostics; Mannheim, Germany). In brief, after 72 h floating,
adherent cells were harvested using trypsine-EDTA solution after PBS washing. The
cell suspension was spun down and the cell pellet was resuspended in 0.1 ml of a marker
solution (2 μl Annexin-V-Fluos (50 × concentrated; Roche) in HEPES buffer (10 mM HEPES,
140 mM NaCl, 5 mM CaCl2; pH 7.4) containing 2 μl of a 50 μg ml−1 PI stock solution).
The suspension was incubated for 15 min in the dark; afterwards, 0.2 ml HEPES buffer
were added and kept on ice until further processing. Analysis of cell size and fluorescence
intensity was performed flow cytometrically on the FACScalibur. After exclusion of
necrotic debris, apoptotic and nonapoptotic (viable) cells were assessed.
RESULTS
Drug sensitivity in normoxic conditions and relative effect of hypoxia
The drug sensitivity was assessed by the MTT assay under normoxic (20% O2) and hypoxic
(1% O2) conditions for 72 h. The results are summarised in Table 1
Table 1
Mean values of IC50 in normoxia and hypoxia (±standard deviation) of cytotoxic drugs
after 72 h in culture of embryonal carcinoma (EC)-derived cell lines (NT2, 2102 EP,
and NCCIT)
NT2
2102 EP
NCCIT
Average
Average
Average
Agent
Normoxia IC50 (±s.d.)
Hypoxia IC50 (±s.d.)
Relative increase in IC50 (H : N) (±s.d.)
Normoxia IC50 (±s.d.)
Hypoxia IC50 (±s.d.)
Relative increase in IC50 (H : N) (±s.d.)
Normoxia IC50 (±s.d.)
Hypoxia IC50 (±s.d.)
Relative increase in IC50 (H : N) (±s.d.)
Cisplatin
0.42 (±0.12)
0.83 (±0.25)
2 (±0)
0.8 (±0.04)
4.1 (±0.42)
5 (±0.28)
1.7 (±0.59)
12 (±3.2)
8 (±1.2)
Oxaliplatin
1.4 (±0.23)
3.8 (±0.15)
2.9 (±0.55)
1.3 (±0.09)
47 (±0.28)
35 (±2.6)
2.6 (±1.39)
62 (±25)
26 (±7.7)
Carboplatin
2.95 (±0.48)
14 (±4.04)
4.7 (±0.55)
6 (±2.55)
>100 (±n.d.)
>12 (±>3.5)
12 (±1.84)
>100 (±n.d.)
>8 (±>1.1)
Gemcitabine
0.055 (±0.044)
0.09 (±0.08)
1.6 (±0.08)
0.53 (±0.08)
>100 (±n.d.)
>100 (±n.d.)
0.9 (±0.09)
>100 (±n.d.)
>100 (±n.d.)
Etopophos
0.16 (±0.1)
0.18 (±0.1)
1.1 (±0.07)
0.2 (±0.15)
>100 (±n.d.)
>100 (±n.d.)
0.36 (±0.06)
>100 (±n.d.)
>100 (±n.d.)
Bleomycin
0.11 (±0.014)
2.1 (±0.39)
19 (±1.1)
0.16 (±0)
>75 (±>35)
>100 (±n.d.)
1.1 (±0.07)
>100 (±n.d.)
>95 (±>7)
Mitomycin C
0.04 (±0.014)
0.06 (±0.01
1.6 (±0.21)
0.4 (±0)
9 (±0.92)
23 (±2.3)
0.59 (±0.06)
7 (±0.21)
12 (±1.5)
Irinotecan
0.43 (±0.082)
0.74 (±0.08)
1.7 (±0.38)
1.3 (±0.08)
>100 (±n.d.)
>79 (±>2.1)
1.8 (±0.49)
44 (±9)
25 (±1.6)
Ifosfamide
2.85 (±1.2)
3.6 (±1.3)
1.3 (±0.11)
5.2 (±1.1)
80 (±23)
15 (±1.1)
7 (±1.1)
34 (±6)
4.7 (±0.04)
Paclitaxel
0.0035 (±6.08 E-4)
0.004 (±7.4 E-4)
1.1 (±0.07)
0.0038 (±1.53 E-4)
>83 (±>29)
>100 (±n.d.)
0.0043 (±7.1 E-5)
88 (±0.71)
>100 (±n.d.)
Additionally, the relative increase (±s.d.) of IC50 in hypoxia compared to normoxia
(H : N) is listed. Values in μ
M. N – normoxia. H – hypoxia. s.d. – standard deviation. n.d. – not defined.
.
Under normoxic conditions, the sensitivity towards cisplatin of the different cell
lines varied by factor 4 at the IC50 values. Carboplatin showed the least cytotoxicity
on an equimolar basis of all drugs tested in normoxia. In contrast, for oxaliplatin,
the IC50-values varied only by a factor 2 in the three EC-derived cell lines. Apart
from cisplatin, paclitaxel showed the highest activity in the three cell lines. No
correlation was found between the sensitivity to paclitaxel and that to cisplatin
under normoxic conditions.
Under hypoxic conditions, all drugs tested were less effective (Table 1), including
mitomycin C (eg, increase of IC50 in normoxia compared to hypoxia for NT2: 1.7-fold
increase, Figure 1A
Figure 1
Cell viability, expressed as percent of the control (%means±s.d., which is indicated
by the bars of the line plots) of EC cells in culture after 72 h. (A) NT2 cells treated
with mitomycin C. (B) 2102 EP cells treated with cisplatin. Note that both drugs,
mitomycin C and cisplatin, are less effective in hypoxia.
, and 2102 EP: 25-fold increase) and cisplatin (eg, NT2: two-fold increase, and 2102
EP: five-fold increase, Figure 1B). For mitomycin C, experimental modification of
the extracellular pH to 6.5 did not result in an enhanced activity in NT2 and NCCIT
cells during hypoxia (data not shown).
The relative effect of hypoxia on the IC50 depended strongly on the cell line. NT2
cells showed a minor effect in chemosensitivity in hypoxia vs normoxia (eg, etopophos:
1.1-fold increase), 2102 EP cells exhibited overall a stronger effect of hypoxia (eg,
etopophos: >100-fold increase). Additionally, the effect depended only to a restricted
extent on the drug, but more clearly on the cell line used (eg, NT2/paclitaxel: 1.2-fold
increase with an IC50 in normoxia: 3.1 nM, and hypoxia: 3.7 nM; Figure 2A
Figure 2
Cell viability, expressed as percent of the control (%means±s.d., which is indicated
by the bars of the line plots) of EC cells in culture after 72 h treated with paclitaxel.
(A) NT2 cells. (B) NCCIT cells.
, and NCCIT/paclitaxel: >100-fold increase with an IC50 in normoxia: 4.3 nM, and hypoxia:
87 μ
M; Figure 2B).
Cell proliferation in normoxia vs hypoxia
In normoxia, doubling times of the three cell lines were 23, 25, and 35 h for NCCIT,
NT2, and 2102 EP, respectively. Under hypoxic conditions, NT2 cells showed a slower
cell growth requiring 36 h for cell doubling. 2102 EP and NCCIT stopped growing under
hypoxic conditions. In 2102 EP, the cell number dropped by approximately 51% and in
NCCIT by 4%. The reduced growth rate was correlated with chemosensitivity of the TGCT
cell lines under hypoxic conditions in vitro (see Table 1). However, it did not strictly
correlate with the relative resistance to all drugs. For example, 2102 EP cells treated
with gemcitabine (Figure 3B
Figure 3
Cell growth, expressed as optical density at 570 nm (ODmeans±s.d., which is indicated
by the bars of the line plots) of EC cells in culture treated with gemcitabine after
72 h. (A) NT2 cells. (B) 2102 EP cells. Note the reduced cell growth rate in hypoxia,
which did not correlate with drug resistance. OD – optical density. nm – nanometers.
) displayed an average increase of IC50 in hypoxia >100-fold compared to normoxia
and five-fold for cisplatin (see Figure 1B). Compared to 2102 EP cells, NCCIT cells
showed an improved survival in hypoxia, but they also indicated an average increase
of IC50 in hypoxia compared to normoxia for gemcitabine >100-fold, and eight-fold
for cisplatin. In contrast, NT2 treated with gemcitabine displayed a similar cell
expansion under normoxic and hypoxic conditions (Figure 3A), with an average increase
of 1.2-fold.
Flow-cytometric analysis of the cell cycle progression revealed that hypoxic conditions
induced a G1 arrest for NT2 and NCCIT cells (Figure 4B and D
Figure 4
Histogram plots of the cell cycle analysis of NT2 (A) and NCCIT cells (C) in normoxia
and NT2 (B) and NCCIT (D) in hypoxia after 48 h by flow-cytometric staining with PI.
M1 – G1 phase. M2 – G2 phase. M3 – apoptotic cells.
) after 48 h, while under normoxic conditions no cell cycle phase synchronisation
occurred (Figure 4A and C). Additionally, Figure 4B shows apoptosis (leakage of fragmented
DNA from apoptotic nuclei; fraction M3) induced by hypoxia in NT2 cells compared to
the cell line NCCIT (Figure 4D).
After 72 h in normoxia, the pH of the medium dropped from pH 7.8 to 6.7 for NT2 and
from pH 7.6 to 6.4 for NCCIT. In hypoxia, the pH of the medium dropped only from pH
7.7 to 7.6 for NT2 and from pH 7.5 to 7.3 for NCCIT in the same time span.
Induction of apoptosis
To evaluate the achieved differences of drug susceptibility of the TGCT cells in normoxia
and hypoxia, the results from the colorimetric MTT assay were verified by flow cytometry.
Viable cells (exclusion of PI and Annexin) and cells killed by cisplatin and paclitaxel
(exclusion of PI, binding of Annexin) after a 72 h drug exposure of NT2 and 2102 EP
were analysed by quantitating PI/Annexin-V labelling. The flow-cytometric results
confirmed that cisplatin and paclitaxel are more effective in normoxia. For cisplatin,
the relative resistance increased two-fold as measured by MTT, and 3.6-fold as assessed
by FACS in NT2 cells in hypoxia compared to normoxia.
DISCUSSION
In this in vitro study, three different TGCT cell lines were used to investigate the
efficacy of several cytotoxic agents. The cell lines differed in their relative sensitivity
to cisplatin by factor of 4. For oxaliplatin, the activity was almost similar in NT2
(cisplatin-sensitive) and 2102 EP (cisplatin-resistant) cells, and increased only
by a factor 2 in NCCIT (cisplatin-resistant) cells. These in vitro data corroborate
our previous clinical data describing a palliative oxaliplatin-based treatment option
in patients with cisplatin-refractory germ cell cancer (Kollmannsberger et al, 2002).
Carboplatin showed cross-resistance to cisplatin and a markedly lower activity on
an equimolar level. Among the various agents used in this study, paclitaxel was very
active in all cells, with no relative increase in IC50 values in cells where cisplatin
was clearly less active. In line with this finding, Motzer et al (1995) described
a marked efficacy of this drug in a teratocarcinoma cell line, particularly in cisplatin-resistant
cells.
The main objective of this in vitro study was to investigate the relative efficacy
of several chemotherapeutic agents used in the treatment for metastatic TGCTs during
normoxic and hypoxic conditions. The oxygen content used in hypoxia models ranges
from <0.1 to 1%. Culturing of the different GCT-derived cell lines in an atmosphere
containing 1% oxygen induced a growth arrest and, in case of NT2 and 2102 EP cells,
also cell deaths. Therefore, lowering the oxygen content further would have precluded
a meaningful analysis due to lack of viable cells. To our knowledge, there are no
data on the physiologic oxygen content in primary TGCTs or in metastases. Frequently
encountered widespread necrotic areas suggest an insufficient blood supply and consequently
hypoxia at least in some areas of these tumours.
Hypoxia has been shown to induce resistance against various agents and radiation (Brown
and Giaccia, 1998; Hoeckel and Vaupel, 2001; Koukourakis et al, 2001; Vaupel et al,
2001). Conflicting data have been described for cisplatin, the most active drug in
the treatment of TGCTs (Liang, 1996; Skov et al, 1998; Kovacs et al, 1999). The impact
of hypoxia on the efficacy of the chemotherapeutic agents cisplatin, etoposide, bleomycin,
ifosfamide, and carboplatin, all used in standard chemotherapy regimens for TGCTs,
and of paclitaxel, gemcitabine, and oxaliplatin, drugs now used in patients with cisplatin-refractory
disease (Bokemeyer et al, 1996; Motzer et al, 2000; Einhorn, 2002; Kollmannsberger
et al, 2002; Shelley et al, 2002) was studied in three different cell lines. All drugs
were less effective under hypoxic conditions. In contrast to data obtained from other
tumour entities (Kennedy et al, 1983; Luk et al, 1990; Yamagata et al, 1992; Sanna
and Rofstad, 1994; Liang, 1996; Skov et al, 1998; Kovacs et al, 1999), this also held
true for the use of cisplatin and mitomycin C in GCTs.
Particularly for mitomycin C, this finding is unexpected. Mitomycin C has been postulated
to be an alkylating agent requiring reduction for activity, and anaerobic conditions
enhance the cytotoxicity (Iyer and Szybalski, 1964). Rockwell (1986) showed that the
cytotoxic effects of mitomycin C increased at acidic pH culture conditions in vitro.
At a low pH (6.0–7.0), mitomycin C can be spontaneously reduced to an alkylating species
without enzymatic activation, while, in the physiologic pH range (7.0–7.4), the cytotoxic
effect of mitomycin C does not vary with the pH (Rockwell, 1986). In our system, the
pH of the medium of untreated cells under hypoxia was in the physiologic range and
did not change after 72 h, probably due to the slower growth of NT2 cells or the growth
arrest of NCCIT cells in hypoxia. Compared to that, the pH of the medium of untreated
normoxic cells decreased to acidic pH values between 6.0 and 7.0. However, mitomycin
C was also less effective in hypoxia at an experimentally acidified pH (6.5). Furthermore,
this study demonstrates that mitomycin C already exhibited a significant cytotoxic
effect in TGCT cells during hypoxia with 1% pO2 in vitro. In contrast to these results,
Kennedy et al (1980) and Teicher et al (1981) achieved a selective toxicity of mitomycin
C in mouse mammary tumour cells using considerable lower (<0.1%) oxygen tensions prior
to the addition of the drug. As mitomycin C acts in a cell cycle-dependent manner,
the pronounced effect of hypoxia on proliferation and the observed G1/S arrest might
prevail the bioreductive activation in our model.
These findings may also serve as a rationale for clinical studies on tumour oxygenation
and response to chemotherapy in GCT patients. A previous retrospective analysis of
haemoglobin values at the end of treatment and prognosis in GCT patients undergoing
sequential dose intensive chemotherapy has indicated that patients with a haemoglobin
level <10.5 g dl−1 postchemotherapy may have a significantly inferior outcome (Bokemeyer
et al, 2002). Tumour oxygenation depends, among other factors, on the haemoglobin
content of the blood. Hence, correction of tumour-associated anaemia – for example,
with recombinant erythropoietin – may improve the pO2 in tumour tissue. The use of
erythropoietin in anaemic cancer patients has been studied to reduce the need for
transfusions and to improve the quality of life (QOL). In patients with head and neck
tumours receiving erythropoietin, an improved outcome of treatment has also been suggested.
Based on the results presented, the hypothesis should be tested as to whether raising
the haemoglobin level in patients with GCTs undergoing chemotherapy might improve
the treatment outcome.
The presented data also allow for some conclusions regarding the mechanisms involved
in the relative drug resistance induced by hypoxia. The impact of hypoxia on chemosensitivity
depended strongly on the cell line. The least effect was evident in NT2, the only
cells that kept proliferating under hypoxic conditions. The two remaining cell lines
showed a far more pronounced relative drug resistance in hypoxia. These findings allow
for two different interpretations: NT2 could be less sensitive for the effect of hypoxia
in general, that is, hypoxia-induced effects are less pronounced. Alternatively, despite
similar changes in hypoxia-induced gene expression overall, only the effect on cell
proliferation differs between the cells. The latter interpretation would point to
the effect on proliferation as the main factor determining the relative effect of
hypoxia on drug sensitivity. However, there was no strict correlation between cell
proliferation and cytotoxic effect indicating relevant influences of factors other
than proliferation.
The hypoxia induced relative resistance to cytotoxic agents depended only partly on
the specific substance. Despite the different modes of action – for example, for gemcitabine
introduction of single-strand DNA breaks, and for etoposide and irinotecan topoisomerase
inhibition – the relative increase in resistance to these drugs during hypoxia was
similar. So far, the potential relevant resistance mechanisms for some of the drugs
with similar behaviour under hypoxic and normoxic conditions have been considered
to be nonoverlapping. Of the substances tested, only etoposide is transported out
of the cells by P-glycoprotein (P-gp). Paclitaxel – a stabiliser of β-microtubulin
polymerisation disrupting the formation of the normal mitotic spindles and thereby
blocking mitosis (Horwitz, 1992) – is independent of P-gp (Lautier et al, 1996). Therefore,
a HIF-1α-mediated induction of P-gp under hypoxic conditions – as recently proposed
by Wartenberg et al (2003) – can be ruled out as a dominating resistance mechanism
in hypoxia in our setting. Bleomycin causes DNA breaks through direct binding to DNA.
This process depends on oxygen and produces reactive oxidative species (ROS), which
may also play a role in the toxicity of bleomycin (Sikic, 1986). P53 does not seem
to play an essential role in drug resistance under hypoxic conditions in the models
chosen here, as NCCIT cells express mutant p53, and NT2 and 2102 EP express wild-type
p53 (Burger et al, 1997). The broad spectrum of substances with unrelated modes of
action and potential means of resistance suggests that rather universally active mechanisms
or coactivation of several pathways confer resistance under hypoxic condition. Kinoshita
et al (2001) reported that cancer cells might obtain resistance to apoptosis once
they have survived hypoxia. The underlying mechanism remains elusive so far. Other
investigators have also suggested that tumour cells acquire antiapoptotic features
and will be selected by hypoxia (Kim et al, 1997).
In summary, this extensive in vitro study using several cytotoxic drugs in three TGC
tumour cell lines shows the importance of normoxic conditions regarding treatment
sensitivity in this tumour model for all chemotherapy agents investigated.