Cancer of the exocrine pancreas is the fourth leading cause of cancer-related deaths
in both men and women (Fernandez et al, 1994). Even if advances in surgical techniques,
radiation and chemotherapy have provided significant improvements in the overall survival
and on the quality of life, fewer than 5% of pancreatic cancer (PC) patients survive
5 years after diagnosis (Parker et al, 1996). Therefore, it is not surprising that
PC is a serious health problem. Thus, there is a great demand for new and more effective
therapeutic strategies for the treatment of this disease. Recent studies on pancreatic
tumour tissues and cell lines have shown that multiple subsets of genes undergo activation
or inactivation during development and progression of disease (Hilgers and Kern, 1999).
Specific point mutations at codon 12 of the K-ras oncogene are detected in 75–90%
of PC specimens and constitute the most common genetic changes in PC (Almoguera et
al, 1988). The mutated K-ras protein is not able to convert GTP to GDP, resulting
in constitutively active GTP-bound species and constitutive activation of cell proliferative
signals. It is therefore conceivable that the pancreatic carcinogenetic process and/or
the maintenance of the transformed phenotype strongly rely on the dysregulated activity
of the p21ras-mediated signalling. The inhibition of p21ras signalling has therefore
been postulated as a possible target for anticancer therapy and might be specifically
suitable for the treatment of PC.
Bisphosphonates (BPs), the most effective and widely used class of drugs in the treatment
of metabolic bone disorders, including tumour-associated bone disease, are capable
of inhibiting p21ras signalling (Luckman et al, 1998; Green, 2001). Therefore, the
extensive evaluation of these compounds, and particularly the study of the recently
available and more potent nitrogen-containing BPs, may suggest expanding applications
of these drugs and warrant further investigation. It is well known that BPs are compounds
that strongly bind to hydroxyapatite crystals and accumulate in mineralised bone (Jung
et al, 1973). Bisphosphonates, which are specifically internalised into osteoclasts
(Sato et al, 1991), lead to inhibition of cell function because of changes in the
cytoskeleton and loss of the ruffled border (Carano et al, 1990), as well as apoptosis
(Hughes et al, 1995). Third-generation, nitrogen-containing BPs, such as zoledronic
acid (ZOL) (Green et al, 1994), inhibit farnesyldiphosphate (FPP) synthase, an enzyme
involved in the mevalonate pathway (Luckman et al, 1998; Benford et al, 1999), preventing
post-translational events of prenylation of small GTP-binding proteins such as p21ras,
Rab, Rho, Rac and cdc42 (Luckman et al, 1998), which are required for a variety of
biological functions including signal transduction and cell adhesion.
In addition to the potent antiresorptive effects of nitrogen-containing BPs, recent
reports have also shown that these compounds induce antiproliferative and apoptotic
effects in multiple myeloma cells in vitro (Shipman et al, 1997; Aparicio et al, 1998),
may synergise with chemotherapeutic or biological agents (Tassone et al, 2000, 2002),
and may offer clinical benefits (Dhodapkar et al, 1998; Berenson et al, 1998). A direct
effect induced by BPs has also been demonstrated on tumour cells of nonhaematopoietic
origin. Bisphosphonates induce inhibition of adhesion of breast and prostate cancer
cells to bone matrix (Van Der et al, 1996; Boissier et al, 1997). In a mouse model
of breast cancer, BPs inhibited the progression and the development of bone metastasis
(Sasaki et al, 1995). Other studies have also suggested that BPs may interfere with
the growth and survival of metastatic cancer cells in bone (Pelger et al, 1998). More
recently, a variety of studies have focused on the direct effect of BPs on growth
and survival of cancer cells from solid tumours. ZOL induces a significant reduction
of cell viability, increases apoptotic cell death and downregulation of bcl-2 protein,
p21ras delocalisation from the cell membrane and proteolytic cleavage of PARP, indicating
direct antitumour effects on human breast cancer cells (Senaratne et al, 2000; Senaratne
et al, 2002). In prostate cancer cells, ZOL has shown a remarkable inhibitory effect
on cell proliferation by induction of cell death and/or cytostasis in vitro (Lee et
al, 2001).
In this scenario, since there have been no studies addressing the possibility of a
direct effect of BPs on growth and survival of human PC cells, we studied the activity
of the most potent BP, ZOL, on a panel of different human PC cell lines (BxPC-3, CFPAC-1
and PANC-1). We have additionally studied whether the nitrogen-containing ZOL might
induce effects on the p21ras/raf1/MEK1/ERK and on the pkB/Akt pathways, and if apoptosis
should be related to caspase-9/-3 and PARP cleavage/activation in these cancer cells.
MATERIALS AND METHODS
Cell cultures and reagents
The PC cell lines BxPC-3, CFPAC-1 and PANC-1 were purchased from the American Type
Culture Collection (Rockville, MD, USA). BxPC-3 and CFPAC-1 are adherent human pancreatic
adenocarcinoma cell lines that were grown in RPMI 1640 and Iscove's modified Dulbecco's
media, respectively. PANC-1 is a human epithelioid pancreas carcinoma cell line that
was grown in Dulbecco's modified Eagle's medium (DMEM). All media were supplemented
with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 μg ml−1 streptomycin
and 100 U ml−1 penicillin. All PC cell lines were cultured at a constant temperature
of 37°C in a humidified atmosphere of 5% carbon dioxide (CO2). The bisphosphonic acid
monohydrate ZOL, 1-hydroxy-2-[(1H-imidazol-1-yl) ethylidene], a third-generation BP,
was kindly provided as the hydrated disodium salt by Novartis Pharma AG (Basel, Switzerland).
The neutralised sodium salt of ZOL was dissolved in sterile ddH2O and used at a final
concentration in a range of 1–100 μ
M. Stock solutions of ZOL were aliquoted and kept at −20°C for long-term storage.
Anti-caspase-9/-3 and anti-PARP MAbs were purchased from New England Biolabs (Beverly,
CA, USA). Protein sepharose was purchased from Sigma (St Louis, MO, USA). Rabbit antisera
raised against raf-1 C-12, βactin, ERK-1 K-23 and ERK-2 MAb C-14 were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-pan-ras MAb clone 10 was purchased
from Calbiochem (San Diego, CA, USA). Anti-Akt MAbs, GSK3β fusion protein and anti-pGSK3β
MAbs were all from Cell Signaling Tech. (Beverly, MA, USA).
Cell proliferation assay
Analysis of cell proliferation was performed on PC cells in the presence of increasing
concentrations of ZOL by the MTT assay. Briefly, PC cells (3 × 104 well−1) were seeded
in 96-well plates in serum-containing media and allowed to attach for 24 h. The medium
was then removed and replaced with new medium containing ZOL at different concentrations.
Cells were incubated under these conditions for a time course spanning 72 h. Then
cells were incubated with 10 μl well−1 of thiazolyl blue (MTT, 5 mg ml−1, Sigma) for
1 h at 37°C. After incubation, 100 μl of 0.04 N HCl in isopropanol was added into
each well and the absorbance was measured at a wavelength of 620 nm in a microplate
reader. A value of 100% was assigned to untreated control cultures, and the IC50 was
defined as the concentration of drug that reduced the number of viable cells to 50%
of the control cultures after 72 h of exposure.
Flow cytometric analysis of apoptosis
Apoptotic cell death was analysed by Annexin-V–FITC staining and by propidium iodide
(PI) detection systems. Annexin-V–FITC binds to phosphatidylserine residues, which
are translocated from the inner to the outer leaflet of the plasma membrane during
the early stages of apoptosis. Labelling of apoptotic cells was performed using an
Annexin-V kit (MedSystems Diagnostics, Vienna, Austria). Briefly, cells were incubated
with Annexin-V–FITC in a binding buffer (provided by the manufacturer) for 10 min
at room temperature, washed and resuspended in the same buffer as described by the
manufacturer. Analysis of apoptotic cells was performed by flow cytometry (FACScan,
Becton Dickinson). Propidium iodide analysis of apoptosis was performed using a commercial
kit (MedSystems Diagnostics, Vienna, Austria). The cells were washed in PBS, resuspended
in 190 μl of prediluted binding buffer (1 : 4) and incubated for 10 min with 10 μl
of the 20 μg ml−1 propidium iodide stock solution, and then the apoptotic cells were
analysed by FACScan flow cytometer. For each sample, 2 × 104 events were acquired.
Analysis was carried out by triplicate determination on at least three separate experiments.
Induction of apoptotic cell death was further performed in the presence or absence
of specific caspase inhibitors: VAD-fmk(-fluoromethylketone) which is a caspase-9
inhibitor, VEID-fmk which is specific for caspase-6 and DEVD-fmk which is a caspase-3
inhibitor.
Immunoblotting for p21ras, raf-1, ERK, pERK, caspase-9/-3, Akt
For extract preparation, the cells were washed twice with ice-cold PBS/BSA, scraped
and centrifuged for 30 min at 4°C in 1 ml of lysis buffer (1% Triton, 0.5% sodium
deoxycholate, 0.1N NaCl, 1 mM EDTA, pH 7.5, 10 mM Na2HPO4, pH 7.4, 10 mM phenylmethylsulphonyl
fluoride (PMSF), 25 mM benzamidin, 1 mM leupeptin, 0.025 U ml−1 aprotinin). Equal
amounts of cell proteins were separated by SDS–PAGE. The proteins on the gels were
electrotransferred to nitrocellulose and reacted with the different MAbs. Nitrocellulose
filters were subsequently exposed to HRP-conjugated secondary antibodies. Finally,
the film was washed with PBS/0.05% Tween 20 and immunoreactive proteins were detected
by the ECL, chemiluminescence technique (Amersham, Uppsala, Sweden).
Analysis of pKB/Akt activity
Akt assay was performed using reagents supplied as a kit obtained from Cell Signaling
Technology Inc. (Cell Signaling Tech., Beverly, MA, USA). Briefly, 106 PC cells were
lysed in a buffer containig 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1% Triton X-100, 2.5 mM sodium PPi, 1 mM β-glycerophosphate, 1 mM Na3VO4 and 1 μg ml−1
leupeptin. Equal amounts of lysed proteins were immunoprecipitated overnight with
sepharose-coupled anti-Akt antibodies at 4°C. Immunocomplexes were washed four times
with lysis buffer and twice with kinase buffer composed of 25 mM Tris (pH 7.5), 5 mM
β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4 and 10 mM MgCl2. Immune complexes were
resuspended in 40 μl of kinase buffer, supplemented with 200 μ
M ATP and 1 μg of GSK-3α/β fusion protein, and incubated at 30°C for 30 min. The kinase
reaction was stopped by the addition of 20 μl of 3 × SDS sample buffer. Samples (20 μl)
were analysed by Western blotting with antiphospho-GSK-3α/β antibody after PAGE separation.
Immunofluorescence detection of actin
Pancreatic cancer cells were seeded onto 35-mm culture dishes on sterile coverslips
and allowed to attach for 24 h. Subsequently, the cells were incubated for 48 h in
the presence of ZOL (15 μ
M). The medium was then removed, cells washed with PBS and fixed with 1 ml Cytofix/cytoperm
(Pharmigen, CA, USA) for 45 min at 4°C. Subsequently, the fixation buffer was removed
and cells washed with 1 ml of 1 × washing buffer. For visualisation of filamentous
actin, the cells were exposed to rhodamine–phalloidin (100 μg ml−1) (Sigma) for 10 min
at 4°C and washed with washing buffer. After final washes, coverslips were mounted
on the dishes using a 50% solution of glycerol in PBS. The cells were examined under
a LEICA TCS SP2 confocal microscope.
Statistical analysis
Results are expressed as mean ±s.e. The statistical significance of differences between
the experimental points was analysed using the t-test; differences were considered
significant when P<0.05.
RESULTS
Zoledronic acid induces antiproliferative effects on PC cells
The effect of ZOL on BxPC-3, CFPAC and PANC-1 PC cells was investigated in vitro using
the MTT assay. Treatment with ZOL (1–100 μ
M) produced a dose-dependent reduction of cell growth after 72 h of treatment (Figure
1A
Figure 1
Zoledronic acid inhibits the growth of PC cells. (A) The cell lines BxPC-3, CFPAC-1
and PANC-1 (3 × 104 ml−1) were seeded in 96-well plates and incubated for 24 h. After
24 h, medium was removed and replaced with medium containing increasing concentrations
of ZOL (1–100 μ
M). After 72 h of incubation, cell proliferation was determined by the MTT assay.
A value of 100% was assigned to untreated control cultures, and the antiproliferative
effects of ZOL were calculated as percentage of residual growth. Each point is the
mean value of at least four replicate experiments ±s.e. (B) The IC50 drug concentration
for each cell line was measured by a logarithmic interpolate curve on the percentage
of residual growth at each concentration point (C and D) Morphological changes induced
in cell cultures after 72 h exposure to ZOL. Analysis was performed by inverted phase-contrast
microscopy. (C) Untreated cultures; (D) treated cultures with ZOL (50 μ
M).
) and the IC50 was calculated in a range of 10–50 μ
M (Figure 1B). Figure 1C and D show the morphological changes of cultured PC cells
after 72 h exposure to ZOL (50 μ
M). Untreated cells (Figure 1C) were flat and well spread, but exposure to the drug
(Figure 1D) resulted in significant antiproliferative effects, retraction of cells
from the substratum, rounding up and loss of contact between neighbouring cells. Altogether,
these findings indicate that ZOL exerts growth inhibitory activity on PC cells.
Zoledronic acid induces apoptotic death of PC cells
To clarify the mechanisms of ZOL-induced growth inhibition, we performed apoptotic
assays on PC cells exposed to this compound. Activation of apoptosis was detected
by Annexin-V staining, which is early expressed on the outer side of the cell membrane
only when apoptosis is triggered and by PI which directly measures fragmented DNA.
Figure 2
Figure 2
(A) Apoptotic effects induced by ZOL on CFPAC-1, PANC-1, BxPC-3 PC cancer cells. The
analysis of apoptosis was performed after exposure to 50 μ
M of ZOL by flow cytometric detection of Annexin-V immunostaining. Cells were pulse
exposed for 30, 60 and 180 min or continuously exposed to the drug. The PI method
was used when apoptosis was evaluated 24 h after beginning treatment. Data are expressed
as the mean value of at least four replicate experiments ±s.e. (B) Flow cytometric
profiles from a representative experiment performed on CFPAC-1 cells. The percentage
of apoptotic stained cells (%) is indicated in each quadrant.
shows the apoptotic death of BxPC-3, CFPAC-1 and PANC-1 cells after 6, 12 and 24 h
exposure to 50 μ
M ZOL. Apoptotic cell death was detected in 35–90% of treated PC cells, suggesting
a significant role of apoptotic death in the in vitro activity of ZOL. Notably, induction
of the apoptosis was independent from the length of ZOL exposure. After 6–12 h from
the beginning of ZOL exposure, Annexin-V staining was equally detected in cells treated
by a 30–180 min pulse as compared with cells treated with continuous drug exposure,
suggesting that induction of the apoptotic process is an early event in pancreatic
cells exposed to ZOL. DNA fragmentation demonstrated by the PI experiment became evident
after 24 h and again was independent from the length of exposure (pulse vs continuous).
Caspase- and PARP-dependency of ZOL-induced apoptosis in PC cells
We investigated the molecular mechanisms of ZOL-induced apoptosis in these cells.
We found that exposure of PC cells to ZOL increased PARP cleavage, a key enzyme in
the apoptotic cascade, as demonstrated by Western blot analysis, which showed a higher
expression of the 116 kDa proenzyme and the appearance of an 89 kDa cleavage product
in treated cells. The maximal effect was detected after 72 h of ZOL exposure (Figure
3A
Figure 3
Role of caspase-9/-3 and PARP in apoptotic cell death induced by ZOL. (A) Detection
of caspase-9/-3 and PARP in PC cells after ZOL treatment. Western blotting analysis
was performed on protein extracts from solubilized whole cell pellets from BxPC-3
and PANC1 PC cell lines after 48 h exposure to ZOL (50 μ
M) demonstrating abundant cleaved species of both caspase-9 and PARP in lysates for
drug-exposed cells, while caspase-3 remained almost completely uncleaved. The experiments
were performed at least three times and the results were always similar. (B) Apoptotic
effects induced by ZOL in the presence of caspase inhibitors. Analysis was performed
by flow cytometric immunodetection of Annexin-V staining on BxPC-3 cells after 48 h
exposure to ZOL (50 μ
M). VAD is a caspase-9 inhibitor, VEID is a specific caspase-6 inhibitor and DEVD
is a caspase-3 inhibitor. Pretreatment with 15 μ
M VAD and 15 μ
M VEID significantly antagonised ZOL-induced apoptosis (P<0.005).
). We also found that caspase-9 was cleaved/activated after cell exposure to the drug,
while no effect was detected on caspase-3. These data were confirmed by the use of
specific caspase inhibitors. We demonstrated that the caspase-9 inhibitor Z-VAD and
the specific caspase-6 inhibitor VEID almost completely prevented apoptotic death
of PC cells, while the DEVD caspase-3 inhibitor only slightly affected apoptosis induced
by ZOL, as detected by flow cytometric analysis of Annexin-V staining (Figure 3B).
These findings suggest that apoptosis induced by ZOL on human PC cells is caspase-9-,
caspase-6- and PARP-dependent, but caspase-3-independent.
Zoledronic acid inhibits the p21ras/Raf1/MEK/ERK1-2 mitogenic and pKB/Akt antiapoptotic
pathways in PC cells
In order to investigate the molecular mechanism of ZOL-induced growth inhibitory activity,
we analysed whether the drug would affect the function of the ras/raf1/MEK/ERK1-2
pathway. We observed a strong decrease of the intracellular content of p21ras and
of its substrate Raf-1 as evaluated by Western blotting (Figure 4A and B
Figure 4
Effects of ZOL treatment on p21ras/Raf-1/MEK1/ERK signalling and PK-B/Akt pathways
in PC cells. Western blotting analysis of p21ras (A), raf-1 (B), Erk-1/2 (D) and pErk
(E). The immunoblotting shows downregulation of p21ras (A) and raf-1 (B) levels after
48 h of exposure to 50 μ
M ZOL. While no significant effects were detected on Erk-1/2 expression after exposure
of cells to the drug (D), the active phosphorylated Erk-1/2 (recognised by an anti-pERK
Mab) levels were greatly reduced in ZOL-treated cells (E). (F and G) Akt expression
and functional analysis in BxPC-3, PANC-1 and CFPAC-1 pancreatic adenocarcinoma cells.
For detection of Akt activity, immunoprecipitation was performed from cell lysates
with anti-Akt antibody, and an in vitro kinase assay was performed using GSK3α/β as
a substrate, followed by Western blotting analysis with antiphospho-GSK3α/β. GSK3α/β
phosphorylation by immunoprecipitated Akt was downregulated after 48 h treatment of
PC cells with 50 μ
M ZOL (F), whereas the Akt protein levels remained unmodified. The experiments were
performed at least three times and the results were always similar. CTR: untreated
cells; ZOL: ZOL-treated cells.
). In fact, 48 h treatment with 50 μ
M ZOL induced a two-to-three-fold reduction of the expression of the two signalling
components (Figure 4A and B). Subsequently, we studied the effects of ZOL on the activity
of ERK1-2, the terminal enzyme of the ras/raf-1 antiapoptotic and survival pathway.
We found a strong decrease of the activity of the two enzymes, as demonstrated by
the immunodetection of the phosphorylated forms of ERK1-2 (Figure 4E). On the other
hand, no changes in the intracellular levels of ERK1-2 were found in the ZOL-treated
PC cells (Figure 4D). These data suggest a significant inhibition of the signalling
activity of this mitogenic and survival pathway. Another important and ras-dependent
survival pathway of human cancer cells is mediated by the activation of the intracellular
kinase pKB/Akt. Therefore, we also studied the effects of ZOL on Akt activity. We
observed a significant impairment of pKB/Akt activity in cells exposed to ZOL as demonstrated
by reduced phosphorylation of the GSK3α/β fusion protein induced by the immunoprecipitated
Akt (Figure 4F). We conclude that ZOL interferes with signalling pathways, which mediate
proliferative stimuli and protect tumour cells from triggering apoptosis.
Zoledronic acid induces actin cytoskeletal reorganisation in PC cells
Finally, we studied whether the morphological changes induced in PC cells after exposure
to ZOL occurred together with cytoskeletal reorganisation, which is considered to
antagonise cell migration and invasion (Nobes and Hall, 1995). After 48 h exposure
to ZOL (15 μ
M), we observed reorganisation of the cytoskeleton and cortical actin polymerisation
as judged by confocal microscopy detection of phalloidin-stained actin filaments.
At these concentrations and exposure time, ZOL did not induce increased apoptotic
death as compared with untreated cells. Figure 5
Figure 5
Analysis of cytoskeletal reorganisation by confocal microscopy on PANC-1 PC cells
after 48 h exposure to ZOL (15 μ
M). The figure shows actin architecture rearrangements in cortical rings. The cells
were examined under a confocal microscope at a magnification of × 100. The experiments
were performed at least three times and the results were always similar.
shows cytoskeletal rearrangements into cortical rings after the treatment. These findings
suggest that the drug may trigger apoptotic death of PC cells, at least in part by
interfering with cytoskeletal integrity.
DISCUSSION
In this study, we have demonstrated that the most potent antiresorptive nitrogen-containing
BP ZOL causes antiproliferative effects, perturbation of the p21ras/Raf1/MEK/ERK1-2
mitogenic pathway and pKB/Akt survival signalling, and also induces apoptotic death
of human PC cells in vitro. We have also shown that the apoptotic events induced by
the drug directly involve activation of caspase-9, caspase-6 and PARP. The present
study provides the first evidence that a nitrogen-containing BP can directly interfere
with intracellular mitogenic and survival pathways downstream to p21ras and produce
antitumour effects in cultured PC cells.
Bisphosphonates are an emerging class of drugs mostly used in the palliative care
of cancer patients. These compounds are specific inhibitors of osteoclastic activity
and may significantly reduce skeletal complications, thereby sustaining the quality
of life of cancer patients. There is a growing interest in the possibility that BPs
may also improve survival in these patients. Several properties of BPs have been recently
highlighted. Experimental findings suggest that these drugs may directly act on cancer
cells, either by inhibiting tumour cell invasion or adhesion to bone matrix or by
inducing growth inhibitory and/or apoptotic cell death. This proapoptotic ability
of BPs has been strongly correlated with the specific antiresorptive potency of each
compound. Specifically, it has been demonstrated that ZOL has a direct effect on human
multiple myeloma (Shipman et al, 1997), breast cancer (Senaratne et al, 2000), prostate
cancer (Lee et al, 2001) and melanoma cells (Riebeling et al, 2002).
We found that ZOL induces significant antiproliferative effects in a range of 10–50 μ
M. These data agree with most other in vitro studies showing maximal growth inhibitory
effects of BPs at doses higher than what is persistently achievable in the serum of
patients after treatment. In fact, it has been shown that infusion of Pamidronate,
which is a less potent antiresorptive BP commonly used in palliative treatments, gives
serum concentrations ∼10-fold lower (0.5–8 μ
M) (Daley-Yates et al, 1991; Oiso et al, 1994; Berenson et al, 1997).
It is well known that BPs have a rapid clearance from the blood stream and therefore
only effects induced by short-term exposure have a clinical relevance for extra bone
antitumour activity. We have indeed that induction of apoptotic death in PC cells
occurs after only 30 min pulse exposure and does not require continuous drug exposure.
We think therefore that this latter finding increases the potential translational
fall-out of this study. Moreover, we consider of interest that PC has been reported
to metastasize to bone in approximately 15% of the cases. The low rate of bone involvment
is not unexpected, because only with the recent improvements in diagnosis and treatment
PC patients begin to survive long enough to reach the stage of disease most associated
with distant skeletal lesions. Even if it has not been stated which bone sites may
be specifically involved in metastatic spread, it has been suggested that PC metastasises
to bone in a pattern that predominantly favours the pelvic girdle (Lyons et al, 2001).
These lesions may heavily affect the quality of life of advanced cancer patients and
overall survival. Therefore, our data strengthen the rationale for the use of these
drugs in a palliative treatment of skeletal metastatic disease. It has however to
be underlined that palliative effects on the metastasis at the bone site do not necessarily
involve direct antitumour activity.
The molecular bases of the BP antitumour activity remain to be elucidated. However,
one possible mechanism is based on the ability of BPs to inhibit some of the enzymes
involved in the pathway for cholesterol synthesis. Relevant to our study, dysregulation
of p21ras activity appears to be a critical event for the onset of PC. We have shown
that ZOL interferes with the p21ras/raf-1/MEK1/ERK and pKB/akt signalling cascades,
as p21ras and Raf-1 contents were clearly reduced in PC cells, and the active phosphorylated
species of ERK1-2-, and pKB/akt-mediated phosphorylation of GSKα/β were strongly downregulated
in cells exposed to the drug. These data appear to be of specific interest considering
the widely described growth promoting and antiapoptotic activity of the two p21ras-dependent
raf-1/MEK1/ERK and pKB/Akt pathways, and our study provides, at our knowledge, the
first evidence that ZOL affects the intracellular signalling pathway downstream to
p21ras. Moreover, it has been demonstrated that Akt directly inhibits caspase-9 activity
by phosphorylation and, intriguingly, we demonstrated that in our experimental model
ZOL-induced apoptosis is caspase-9-dependent (Fujita et al, 1999; Zhou et al, 2000).
In fact, we demonstrated that, in parallel with the antiproliferative effects mediated
by the drug, ZOL also induces apoptotic cell death in PC cells. Cell death by an apoptotic
process is a well-described phenomenon that is associated with multiple molecular
events. The caspase family of cysteine proteases are central participants in apoptotic
cell death. Recent studies have highlighted the potential role of caspase-9 as a selective
target for anticancer treatment. It is also believed that chemotherapeutic agent-induced
apoptosis is predominantly accomplished by activation of the mitochondrial pathway
(Wu and Ding, 2002). In fact, blockade of caspase-9 decreases chemotherapeutic agent-induced
mitocondrial-dependent apoptosis. In this context our observations are in agreement
with other studies which reported that ZOL-mediated apoptosis is associated with cytochrome
c release and consequent caspase activation (Senaratne et al, 2002). Caspase-3 is
considered to be involved in the execution phase of apoptosis, when proteolysis of
intracellular substrates is a major event. It has been shown that BP-induced osteoclast
apoptosis is dependent on caspase-3 activation since cell death is prevented by caspase-3
inhibitors, thereby suggesting the key role of these proteases in apoptotic cell death
induced by these drugs (Benford et al, 2001). Caspase-3 has also been involved in
apoptotic death of breast cancer cells exposed to ZOL (Senaratne et al, 2002). In
our study however, we have been unable to find a specific role for caspase-3, which
is not cleaved/activated, and apoptosis was only slightly antagonised by selective
caspase-3 inhibition. Our observations are in accordance with others who found no
correlation between the amount of processing of caspase-9 and effector caspases in
human pancreatic carcinoma (Gerhard et al, 2002). We can therefore hypothesise that
in ZOL-exposed PC cells, a caspase-9- and caspase-6-dependent and caspase-3-independent
pathway is operative, while in other tumour cell systems, as well as in normal osteoclast
cells, execution of apoptosis induced by ZOL may occur by a caspase-3-dependent mechanism.
The possible identification of tissue-specific executioners of apoptosis might be
ideal subjects of investigation, and may have the advantage of enhancing selectivity
in therapeutical intervention.
Activation of caspases during cell death is commonly associated with characteristic
intracellular changes of in vitro cultured cells, including loss of adhesion, actin-cytoskeletal
structural reorganisation into cortical rings, cell rounding and contracting, and
generation of apoptotic bodies. A role for caspase-mediated proteolysis of structural
and adhesion proteins has been suggested in the morphological changes that characterise
apoptotic cell death. After the initial phase of contraction and blebbing, caspase-mediated
cleavage of actin monomers probably induces disassembly of actin filaments (Mashima
et al, 1997; McCarthy et al, 1997). We found that after 48 h of low concentration
ZOL treatment, significant actin-cytoskeletal reorganization occurs in PC cells as
demonstrated by actin staining. These modifications indicate that ZOL induces actin
rearrangements into cortical rings and that these events may drive the cells to the
apoptotic process. This specific effect indicates the intriguing possibility of positive
interactions of BPs with drugs that interfere with actin polymerisation and depolymerisation
such as taxoid or vinca alkaloid agents. A synergistic effect of ZOL with paclitaxel
has in fact been demonstrated in human breast cancer (Jagdev et al, 2001). Recent
reports have also highlighted the role of signal transduction pathways controlled
by the Rho family of small GTPases in regulating the architecture of the actin cytoskeleton
and the morphological changes during apoptotic cell death (Coleman and Olson, 2002).
An intriguing preliminary finding is that the small GTP-binding protein RAC, functionally
related to cytoskeletal organisation, discloses enhanced (10-fold) expression in ZOL-exposed
cancer cells as detected by Western blot analysis (data not shown). Based on these
findings, further studies are presently in progress in order to understand the functional
significance of these observations.
In conclusion, we have demonstrated that PC cells are highly sensitive to ZOL-induced
growth perturbation and induction of apoptosis, which is caspase-9- caspase-6- and
PARP-dependent. Moreover, our experimental findings suggest that inhibition of p21ras/Raf-1/MEK1/ERK
signalling as well as PK-B/Akt inhibition might be relevant for the antitumour effects
of ZOL. Although we have been capable of demonstrating that a short-term exposure
is enough for activation of apoptosis, we think that a pharmacokinetic profile more
relevant to the extra-bone antitumour effects of ZOL should be derived and might involve
entrapping of BPs in lysosomes. An additional topic of current investigation is BP
combination with cytotoxic drugs or selective signal transduction inhibitors.