New fluorescent platinum (II) complexes containing anthracene
derivatives as a carrier ligand : synthesis, characterization and in vitro studies
Marqués Gallego, P.
Citation
Marqués Gallego, P. (2009, September 17). New fluorescent platinum (II) complexes
containing anthracene derivatives as a carrier ligand : synthesis, characterization and in vitro studies. Retrieved from https://hdl.handle.net/1887/13999
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/13999
Note: To cite this publication please use the final published version (if applicable).
7 7 . .
In I n vi v it tr ro o s s t t u u d d i i e e s s t t o o w w a a r r d d s s t t h h e e u u n n d d e e r r s s t t a a n n d d i i n n g g o o f f t t h h e e c c e e l l l l u u l l a a r r pr p ro oc c e e ss s si in ng g o of f c ci is s- -[ [P Pt t( ( ba b ap pd da a) )C Cl l
22]* ] *
Abstract
To investigate whether lysosomes play an important role in the uptake of cis-[Pt(bapda)Cl2], intracellular accumulation in the presence of bafilomycin A1 and monensin has been investigated. Bafilomycin A1 shows no effect on the cytotoxicity or on the intracellular accumulation of cis-[Pt(bapda)Cl2] in both the A2780 and the A2780R cells. In contrast, preincubation with monensin decreases the intracellular accumulation of cis-[Pt(bapda)Cl2] in A2780R cells, and increases the cytotoxic activity of this compound in the cisplatin-resistant cell line. Cellular processing of cis-[Pt(bapda)Cl2] in the A2780 and the A2780R cells after incubation with bafilomycin A1 and monensin has been investigated using fluorescence microscopy. Significant differences were observed in the cellular distribution of cis-[Pt(bapda)Cl2] in monensin- or bafilomycin A1-pretreated cells, as compared to that in untreated cells, were observed.
7.1. Introduction
Lysosomes serve as intermediary compartments that receive metals from influx pathways and store them or even distributes them to other destinations in the cells, and have been found to be important in the cellular processing of cytotoxic agents.1-5 In addition, several studies have suggested that resistant cells exhibit enhanced drug efflux, through mechanisms by which the drugs are sequestered in these intracellular vesicles, from where they are removed by exocytosis.6 More specifically, in the case of cisplatin, there is evidence that cisplatin is exported from the cell via initial sequestration into cellular vesicles of the secretory pathway.7 Fluorescence microscopy studies of a fluorescein-conjugated cisplatin analogue indicated that cisplatin is accumulated in intracellular vesicles, containing the copper efflux transporters ATP7A and ATP7B.3, 7 Additional studies have shown that lysosomes, Golgi apparatus, and secretory compartments are involved in the cisplatin efflux.8 In addition, it has been found that lysosomes also participate in apoptotic and necrotic cell-death pathways,9 and that metals activate these cell death pathways as a result of the lysosomal damage.10, 11 Therefore, it is believed that the sequestration might be a mechanism by which cisplatin triggers apoptosis, causing a direct damage on the lysosomes, with the release of the lysosomal contents into the cytoplasm. Furthermore, a reduced number of lysosomal compartments in cisplatin-resistant cells have been observed,3, 5 which might increase the survival of the resistant cells. Alterations in the lysosomal and exosomal pathways observed in cisplatin- resistant cells might have an influence on the nuclear trafficking of cisplatin, decreasing the DNA-platination of cisplatin in the resistant cells. Therefore, a relationship between lysosomal sequestration and cisplatin resistance has been suggested.1, 5
The antitumor drugs, namely anthracycline, doxorubicin, and adriamycin, have been found to be accumulated in acidic intracellular compartments.10, 12-13 It has been proposed that the pH gradient present between the cytoplasm and the acidic vesicles is responsible for the sequestration of these drugs, which are weak bases, within the acidic compartments. The mechanism behind this sequestration has been simplified to the following: the drug encounters an acidic environment in the lysosomes, where it is converted into a charged form. This form is unable to cross internal membranes, being in this way sequestered within acidic vesicles. Evidences of the pH-dependent accumulation
have been obtained using different inhibitors. It is known that many intracellular membrane vesicles contain an H+-adenosine triphosphatase (vacuolar H+-ATPase), which regulates intravesicular pH. Low concentrations of bafilomycin A1 selectively inhibit the vacuolar H+-ATPase, and several evidences that H+-ATPase has been found to display an important role in the pathway of drug efflux for resistant cells.14 The mechanism for this inhibition is still not elucidated; however, it has been suggested that drug efflux occurs via a pathway where the drugs are sequestered in acidic vesicles. 15 In vitro studies with anthracycline-type antitumor drugs in the presence of monensin (inhibitor of the Na+ H+ exchanger), which dissipates the pH gradients across all membranes, has been reported to diminish drug uptake.16 Furthermore, monensin inhibits the daunorubicin efflux from anthracycline-resistant cells.17 This observation suggests that anthracyclines accumulate in acidic vesicles, and from there they are transported and sequestered by exocytosis.
Moreover, preincubation of the doxorubicin-resistant cells with monensin leads to an increased sensitivity to the drug in the resistant cell line. These results suggest a direct relationship between the intracellular pH gradient and drug resistance.18 In the same studies, preincubation with bafilomycin A1 (which specially block the H+ ATPase in endosomes and lysosomes19) was found to increase adriamycin levels within the nucleus in the resistant-MCF7 cells.18 Taken together it appears that there is a relationship between the drug resistance and the sequestration of the drug in cytoplasmic organelles, and that this sequestration might be dependent on organelle acidification.
As described in chapter 6, cis-[Pt(bapda)Cl2] displays fast accumulation in acidic lysosomes in the A2780R cells after short-time incubation. Similar accumulation in the A2780R cells was observed for the ligand bapda, suggesting that the lysosomal sequestration is not related to the cross-resistance of cis-[Pt(bapda)Cl2] in the A2780R cells. Therefore, it is likely that the sequestration of these compounds in lysosomes is related to a specific uptake pathway. As discussed above, changes in the intracellular pH are associated with changes in the drug distribution and the cellular sensitivity to anticancer drugs. To investigate the relationship between the lysosomal sequestration of cis-[Pt(bapda)Cl2] in the A2780 and the A2780R cells, and the intravesicular pH, several in vitro studies using monensin and bafilomycin A1 have been undertaken. Additionally, control studies regarding the effect of bafilomycin A1 and monensin on the A2780 and
the A2780R cells have been performed. These control studies are important to understand the results obtained with cis-[Pt(bapda)Cl2].
7.2. Experimental Section
7.2.1. Materials and reagents
The cis-[Pt(bapda)2Cl2] and the ligand bapda were synthesized as described in Chapter 5. All other chemicals and solvents were reagent-grade commercial materials, and were used as received. Stock solutions of monensin (2 mM), bafilomycin A1 (10 mM), and acridine orange (17 mM) were prepared in PBS, DMSO, and Mili Q water, respectively. All solutions were stored at -20 °C and diluted with complete medium before use.
A 2 mM stock solution of cisplatin in 40 mM NaCl was prepared and aliquots were stored at -20 °C. Stock solutions of cis-[Pt(bapda)Cl2] and the ligand bapda were freshly prepared in DMF and diluted in medium before use. The DMF concentration during the in vitro studies did not exceed 0.5%, and was found not toxic to the cells.
7.2.2. Cell lines, culture conditions and short-time exposure cytotoxic assay The human ovarian carcinoma cell line A2780 and its cisplatin resistant counterpart A2780R were grown as described in Chapter 3 (section 3.2.1). The cytotoxicity studies of the bapda ligand and cis-[Pt(bapda)Cl2], as compared to cisplatin in the A2780 and the A2780R cells, were performed as described in Chapter 3 (section 3.2.3). The results were analyzed and the pEC50 values (EC50 is the drug concentration that produces 50% of the maximum possible response) were determined with the GraphPad Prism™ analysis software package (Graph- Pad Software, San Diego, USA) using non-linear regression (sigmoidal dose response, variable slope). The resistance factor (RF) was calculated by dividing EC50 in the resistant variant by the value of EC50 in the respective sensitive cell line.
7.2.3. Bafilomycin A1 and monensin effect on the cytotoxic activity
The effect of bafilomycin A1 or monensin in the cytotoxic activity of cis-[Pt(bapda)Cl2] in the A2780 and the A2780R cells has been investigated.
On day one 5000 cells/well were plated in 96-wells flat bottom microtiter plates, and incubated for 24 h at 37 °C, 7% CO2 to allow the cells to adhere. On day 2, the cells were preincubated with bafilomycin A1 (100 nM) or monensin (20 M) for 1 h.
Subsequently, the medium was carefully removed, and cis-[Pt(bapda)Cl2] was added at 10 M final concentration and incubated at 37 °C and 7% CO2 atmosphere for additional 24 h. These experiments were repeated independently to verify reproducibility. On day 3, 50 L of a MTT solution (5 mg/ml in PBS buffer) was added in each well and proceeded as described before (Chapter 3, section 3.2.3). The effect of the inhibitors in the cytotoxic activity of cis-[Pt(bapda)Cl2] was determined by taking inhibitor-free cells as 100%
viable cells.
7.2.4. Determination of intracellular platinum accumulation
Cellular accumulation was determined in both cell lines exposed to cisplatin (cis-[PtCl2(NH3)2]), and cis-[Pt(bapda)Cl2] at the final concentration of 50 μM in complete medium. After 60 min of drug exposure, the medium was removed and the cells were washed twice with ice-cold PBS buffer. The cells were collected, and the pellet was stored at -20 °C until analysis. Immediately after thawing the cells were lysed with 100 μL concentrated nitric acid for 60 min on the water bath at 80 °C. The intracellular platinum concentrations were measured by flameless atomic absorption spectrometry (FAAS). The results were related to the cell number (about 3 million).
7.2.5. Bafilomycin A1 and monensin effect on the intracellular accumulation
The A2780 and the A2780R cells were preincubated for 1 h in complete medium containing bafilomycin A1 (100 nM) or monensin (20 M). Subsequently, the cells were washed twice with PBS and fresh medium containing cis-[Pt(bapda)Cl2] at 50 M final concentration was added and incubated for 1 h. The following steps were performed as described in section 7.2.4.
7.2.6. Fluorescence microscopy in the A2780 and the A2780R cells
For the living cell observations the cells were grown in a 35 mm culture dish to 30-50% confluence in complete medium. Pictures were taken using a microscope (IX81;
Olympus The Netherlands) with ×60 objective (Olympus, The Netherlands). The
temperature of the culture medium was controlled between 36 and 37 °C by an objective heater and a heated ring surrounding the culture chamber. The CO2 atmosphere was kept at 5.2% during imaging. Digital images were taken with a cooled CCD camera (F-View, Olympus The Netherlands). The images were processed using the Cell M software.
7.2.7. Bafilomycin A1 and monensin: effect on cellular processing
For inhibition studies the A2780 and the A2780R cells were preincubated in phenol red-free medium containing bafilomycin A1 (100 nM), or monensin (20 M) for 1 h at 37 °C and 7% CO2 atmosphere previous to the incubation with cis-[Pt(bapda)Cl2].
Subsequently, the cells were washed twice with PBS, and phenol red-free and serum-free medium containing cis-[Pt(bapda)Cl2] (10 M) was added, and incubated for additional 15 min. After the incubation the cells were washed twice with PBS and complete phenol red-free medium was added. To compare the cellular processing of cis-[Pt(bapda)Cl2] in the presence or absence of inhibitors, live imaging of both were performed. To detect the fluorescence signal of bapda ligand and cis-[Pt(bapda)Cl2] a filter for ex 377 nm (exposure time = 10 ms) was used.
LysoTrackerTM Red DND-99 (Molecular Probes, Leiden, The Netherlands) was used to stain the acidic vesicles. LysoTrackerTM Red DND-99 was added to the culture medium in a final concentration of 50 nM for 15 min. After incubation with the dye, the cells were washed twice with PBS, and complete phenol red-free medium was added before imaging. For detecting the fluorescence of LysoTrackerTM Red DND-99, TRITC-filter (ex 573 nm; exposure time = 10 ms) was used.
LysoSensorTM Green DND-189 (Molecular Probes, Leiden, The Netherlands) was used to investigate the pH of the A2780 and the A2780R cells. LysoSensorTM Green DND-189 was added to the culture medium in a final concentration of 1 μM for 15 min.
After incubation with the dye, the cells were washed twice with PBS, and complete phenol red-free medium was added before imaging. For detecting the fluorescence of LysoSensorTM Green DND-189, GFP-filter (ex 480 nm; exposure time = 10 ms) was used.
7.2.8. Vesicular pH modification by bafilomycin A1 and monensin
Acridine-orange is a fluorescent weak base that is used frequently as a probe for acidification of organelles.13, 20 When acridine orange is accumulated in a high concentration in acidic compartments its emission shifts from green to red. The A2780 and the A2780R cells were incubated in complete phenol red-free medium containing acridine-orange (6 M) for 10 min. Subsequently, the medium was carefully removed, and the cells were washed twice with PBS. Fresh complete phenol red-free medium was added and images of both, the A2780 and the A2780R cells were taken using
ex = 575 nm and ex = 480 nm for red and green emission, respectively.
Inhibition studies with bafilomycin A1 or monensin were also monitored. After 1 h incubation with inhibitor (bafilomycin A1 (100 nM) or monensin (20 M)) the cells were incubated with acridine orange (6 M) for 10 min. Cellular imaging was performed as previously described.
7.3. Results and discussion
7.3.1. Cell imaging of the human ovarian carcinoma cells. Control studies (i) Lysosomal pH using LysoSensorTM and LysoTrackerTM dyes
Digital cellular imaging using LysoSensorTM Green DND-189, which exhibits a pH-dependent increase in fluorescence intensity, has been used in several cases to qualitatively visualize the differences in the pH between sensitive and resistant cell lines.2, 10, 21 To establish qualitative differences between the pH of the A2780 and the A2780R cells, labeling of both cell lines with LysoSensorTM Green DND-189 was performed. LysoTrackerTM Red DND-99, which is a pH-independent dye, was chosen to label the acidic lysosomes in both cell lines (Fig. 7.1).
Small differences are observed in the fluorescent intensity between the A2780 and the A2780R cells stained with LysoSensorTM Green DND-189. Both cell lines display a strong fluorescence in the acidic environment, suggesting a slightly acidic cytosolic pH.
Moreover, stronger fluorescence in cellular vesicles is observed, which corresponds to lysosomes, as confirmed by the labeling with LysoTrackerTM Red DND-99.
Fig. 7.1. Images A and C: Cells stained with LysoSensorTM Green DND-189 in the A2780 and the A2780R, respectively. Images B and D: Acidic lysosomes stained with LysoTrackerTM Red DND-99, in the A2780 and the A2780R, respectively.
Interestingly, very different lysosomal distribution in both cell lines is observed.
In addition, no significant differences in the acidic pH of the lysosomes are observed, with intense green fluorescence in both the A2780 and the A2780R lysosomes.
Therefore, qualitatively the lysosomes in the A2780R cells present a similar acidic pH, compared to the sensitive counterpart. This is an important parameter, because it has been reported that lysosomal acidification is essential for regular endocytosis.21 A failure in the acidic pH of the lysosomes might be a sign of a general defect in the endocytosis, which has been found to be related to the cross-resistant to cisplatin in the A2780R cell line.2 Hence, labeling the A2780 and the A2780R cells with LysoSensorTM Green DND-189 has shown that there is no evidence of a defect on the acidification of the lysosomes in our cisplatin-resistant cell line, and therefore endocytosis might be involved in the uptake of cis-[Pt(bapda)Cl2] in both cells, since lysosomal accumulation in both cell lines was observed (see Chapter 6).
(ii) Bafilomycin A1 and monensin effect using acridine orange
As discussed above, in vitro studies in tumor cell lines have shown that bafilomycin A1 and monensin are able to modify the pH of the intracellular acidic compartments, affecting the cellular distribution of fluorescent drugs such as adriamycin.18 A qualitative pH distribution monitored with acridine orange has been performed to investigate the pH alterations in the A2780 and the A2780R triggered by bafilomycin A1 or monensin. Acridine orange is a fluorescent weak base that has been
A C B D
used as a probe for acidification of organelles.20 So, acridine orange (6 M) was incubated for 10 min and visualized under the microscope. Parallel cultures preincubating the cells with bafilomycin A1 or monensin, prior to addition of acridine orange were performed, and the effect of these inhibitors is discussed below.
Fig. 7.2. Cellular distribution of acridine orange in the A2780 human ovarian carcinoma cells (imaging after incubation). (A) phase-contrast image of living A2780 cells; (B) acridine orange fluorescence (green);
(C) accumulation of acridine orange in acidic compartments (red); (D) superimposed representation of acridine orange processing (B and C).
Fig. 7.3. Cellular distribution of acridine orange in the A2780R human ovarian carcinoma cells (imaging after the incubation) (A) phase-contrast image of living A2780R cells; (B) acridine orange fluorescence (green); (C) accumulation of acridine orange in acidic compartments (red); (D) superimposed representation of acridine orange processing (B and C).
C
B
D A
A
C D B
In the absence of inhibitors a red fluorescence in cytoplasmic organelles (Fig. 7.2 and 7.3) in both the A2780 and the A2780R cells treated with acridine orange was observed. This observation indicates the accumulation of acridine orange in acidic compartments. It is important to note that acridine orange does not represent an absolute pH value within these compartments. These results demonstrate that there are no significant differences in the subcellular pH between the drug-resistant and drug-sensitive human ovarian carcinoma cells used in our studies. Nevertheless, the distribution of the acidic organelles in the A2780 and the A2780R cells is clearly different, which is in agreement with the labeling studies using LysoSensorTM Green DND-189 and LysoTrackerTM Red DND-99 (Fig. 7.1).
Preincubation of both cell lines with either bafilomycin A1 or monensin for 1 h was performed in parallel cultures. Subsequently, acridine orange was added to the cells and the changes on the cellular processing were followed using fluorescence microscopy.
It has been reported that the Na+ H+ exchanger monensin dissipates pH gradients across all membranes and alters the vesicular trafficking, while the vacuolar H+ ATPase inhibitor bafilomycin A1 is used to block acidification.18 Moreover, bafilomycin A1
inhibits the acidification of endosomes, lysosomes, and trans-Golgi network differently than monensin and, unlike monensin, does not affect membrane permeability.
Preincubating both cell lines with bafilomycin A1 shows a large effect on the acridine orange distribution (Fig. 7.4). Similar redistribution of acridine orange in the A2780 and the A2780R cells, with a large decrease in the accumulation in acidic compartments (red) and mainly a diffuse distribution across the cytoplasm is observed (Fig. 7.4). In contrast, monensin has no effect on the cellular processing of acridine orange (images not shown), with red fluorescence from high accumulation of the acridine in acidic vesicles as observed in monensin-free cells. It is worthy to note that the accumulation of acridine in acidic compartments remains visible up to 24 h after the incubation.
Fig. 7.4. Cellular processing of acridine orange in (A) bafilomycin A1-treated A2780 cells, and (B) bafilomycin A1-treated A2780R cells. Phase-contrast images are shown on the left side; corresponding acridine orange fluorescent images are in the middle and on the right side.
These observations suggest that acridine orange is sequestered in these compartments, and the pH gradient between the cytoplasm and the lysosomes is not altered by monensin. Nevertheless, it is important to mention that reversible effect of the pH alteration 1 min after removal of monensin from the medium has been reported.22 To investigate the reversibility in the effect of monensin in the pH modification in the A2780 and the A2780R cells, monensin was kept in the medium during acridine orange labeling.
Again, monensin caused no effect on the accumulation of acridine orange in acidic compartments (images not shown). Therefore, the lack of effect described above is unlike to be due to a reversible process. The effect of bafilomycin A1 and monensin on the pH of the acidic vesicles is an important parameter. The equilibrium between the uncharged and charged forms of weak base drugs (i.e. anthracyclines, adriamycin, or doxorubicin), found to accumulate in lysosomes. is pH-dependent; therefore, drug accumulation in acidic vesicles is favored by a pH gradient between the cytoplasm and the acidic compartments, whereas acidification of the cytoplasm and/or alkalinization of the acidic vesicles decrease drug accumulation within these organelles. Therefore, the ability to reserve drug resistance by drugs like bafilomycin A1 or monensin, that alkalinize the pH in the acidic compartments of the endosomal and secretory systems indicates that protonation, sequestration, and secretion are the principal elements of resistance to antitumor drugs of several cancer cell lines.10,13, 14, 22
A
B
7.3.2. Cellular processing of cis-[Pt(bapda)Cl2]
Because cis-[Pt(bapda)Cl2] shows sufficient fluorescent emission in the A2780 and the A2780R living cells (chapter 6), the cellular processing of cis-[Pt(bapda)Cl2] in the A2780 and the A2780R cells preincubated with bafilomycin A1, or monensin using fluorescence microscopy have been performed. The cellular processing of cis [Pt(bapda)Cl2] in both cells lines after preincubation with bafilomycin A1 was performed to investigate whether the lysosomal pH is involved in the sequestration of cis [Pt(bapda)Cl2] in acidic vesicles. The same studies using monensin were performed although monensin has shown not to alter the lysosomal pH under the same conditions (according to acridine orange labeling studies). To allow the comparison, fluorescence microscopy of cis-[Pt(bapda)Cl2] in both cell lines in the absence of inhibitors was performed in parallel cultures.
As clearly shown in Fig. 7.5, preincubating the A2780 cells with bafilomycin A1
and monensin has an effect on the cellular processing of cis-[Pt(bapda)Cl2]. Both inhibitors altered the lysosomal accumulation of cis-[Pt(bapda)Cl2], compared to that of the platinum(II) compound in the absence of inhibitor (Fig. 7.5:A). In the A2780 cells, cis-[Pt(bapda)Cl2] exhibits densely packed fluorescent signals dispersed over the cytoplasm when the cells are preincubated with the inhibitors (Fig. 7.5: B and C).
The same studies in the cisplatin-resistant cell line were performed, displaying changes in the cellular processing of cis-[Pt(bapda)Cl2] (Fig. 7.6). The lysosomal accumulation of cis-[Pt(bapda)Cl2] in the A2780R cells is partly inhibited by bafilomycin A1, with a slightly stronger fluorescence signal within vesicles close to the cell nucleus (red circle in Fig. 7.6:B), assigned in previous cases as acidic lysosomes (Fig. 7.1). This observation suggests that the accumulation of cis-[Pt(bapda)Cl2] does not only depend on the pH gradient between the cytoplasm and the acidic lysosomes, since bafilomycin A1
decreases this gradient as shown in the cellular processing of acridine orange (Fig. 7.4).
Preincubating the A2780R cells with monensin also shows accumulation of cis [Pt(bapda)Cl2] within lysosomes (red circle in Fig. 7.6:C), however, also other vesicles in the cells, containing the platinum compound, are observed.
Fig. 7.5. Images in row A: cellular processing of cis-[Pt(bapda)Cl2] in the A2780 cells. Images in row B:
cellular processing in bafilomycin A1-preincubated A2780 cells. Images in row C: cellular processing in monensin-preincubated A2780 cells. Left images are phase contrast, middle images show accumulation of cis-[Pt(bapda)Cl2]. Superimposed phase contrast and fluorescence images are shown on the right.
Fig. 7.6. Images in row A: cellular processing of cis-[Pt(bapda)Cl2] in the A2780R cells. Images in row B:
cellular processing in bafilomycin A1-preincubated A2780R cells. Images in row C: cellular processing in monensin-preincubated A2780R cells. Left images are phase contrast, middle images show accumulation of cis-[Pt(bapda)Cl2]. Superimposed phase contrast and fluorescence images are shown on the right.
A
B
C
A
B
C
The cells were kept at 37 °C in a 7% CO2 atmosphere to follow the cellular processing of cis-[Pt(bapda)Cl2] 24 h after the incubation. Inhibition studies of the H+-ATPase with bafilomycin A1 in the human ovarian carcinoma cells 24 h after the incubation show no significant differences in the cellular processing of the platinum(II) compound compared to inhibitor-free studies (images not shown), with accumulation in acidic lysosomes.
In contrast to the cellular processing in bafilomycin A1-treated cells, large differences have been observed for the cellular processing of the platinum(II) compound in the monensin-preincubated human ovarian carcinoma A2780 and the A2780R cells (Fig. 7.7 and 7.8). The fluorescent signals for cis-[Pt(bapda)Cl2] in both cell lines were partially localized with LysoTrackerTM Red DND-99 dye; however, additional vesicles containing the platinum complex were observed in the cytoplasm.
Preincubating the cells with bafilomycin A1 and monensin was also studied towards the cellular processing of the ligand bapda. The cellular distribution of bapda in the A2780 cells has been described in chapter 6, finding lysosomal sequestration after several hours in the A2780 cells. As shown in Fig. 7.9:A, just after incubation the ligand bapda is not specifically accumulated within the cells, undergoing the same processing in monensin- treated cells (Fig.7.9:C). Interestingly, changes are observed in the cellular processing of bapda when the A2780 cells are preincubated with bafilomycin A1 (Fig. 7.9:B). Stronger fluorescent signals from the ligand bapda are observed close to the cell nucleus.
The alteration in the intracellular pH of the A2780 cells by bafilomycin A1 may be responsible for these changes. When the same studies are performed in the cisplatin- resistant cells an accumulation of bapda in acidic lysosomes is observed (Fig.7.10:A).
When the cells are preincubated with bafilomycin A1 or monensin, non-specific differences are found (Fig. 7.10:B and C) compared to inhibitor-free cells (Fig. 7.10:A).
Moreover, 24 h after the incubation fluorescence signals remained visible and localization studies confirmed that the ligand bapda is accumulated in acidic lysosomes (images not shown).
Fig. 7.7. Cellular distribution of cis-[Pt(bapda)Cl2] in the A2780 cells 24 h after incubation with monensin, and staining with LysoTrackerTM Red DND-99: (A) phase-contrast image of living A2780; (B) cis- [Pt(bapda)Cl2] fluorescence (blue); (C) staining of lysosomes (red); (D) superimposed representation of the images of cis-[Pt(bapda)Cl2] (blue) and LysoTrackerTM Red DND-99 (red).
Fig. 7.8. Cellular distribution of cis-[Pt(bapda)Cl2] in the A2780R cells 24 h after incubation with monensin, and staining with LysoTrackerTM Red DND-99: (A) phase-contrast image of living A2780R cells; (B) cis-[Pt(bapda)Cl2] fluorescence (blue); (C) staining of lysosomes (red); (D) superimposed representation of the images of cis-[Pt(bapda)Cl2] (blue) and LysoTrackerTM Red DND-99 (red).
A
C D B
A
E C
B
Fig. 7. 9. Images in row A: cellular processing of ligand bapda in the A2780 cells. Images in row B:
cellular processing of ligand bapda in bafilomycin A1-preincubated A2780 cells. Images in row C: cellular processing of ligand bapda in monensin-preincubated A2780 cells.Left images are phase contrast, middle images show accumulation of cis-[Pt(bapda)Cl2]. Superimposed phase contrast and fluorescence images are shown on the right.
Fig. 7.10. Images in row A: cellular processing of ligand bapda in the A2780R cells. Images in row B:
cellular processing of ligand bapda in bafilomycin A1-preincubated A2780R cells. Images in row C:
cellular processing of ligand bapda in monensin-preincubated A2780R cells. Left images are phase contrast, middle images show accumulation of cis-[Pt(bapda)Cl2]. Superimposed phase contrast and fluorescence images are shown on the right.
A
B
C
A
B
C
7.3.3. Cytotoxic activity in short-time exposure experiments
To investigate the relationship between the cytotoxicity of this new compound and its ability to pass the cell membrane and accumulate within the cell, short-exposure cytotoxic activity measurements of cis-[Pt(bapda)Cl2] were performed. In addition, short- time exposure cytotoxic studies allow the recovery of the cells during the post-incubation time with drug-free medium, establishing in this way whether cis-[Pt(bapda)Cl2] is highly toxic to the cells. The results of short-time exposure experiments for cis-[Pt(bapda)Cl2] as compared to cisplatin are summarized in Table 7.1.
Table 7.1. pEC50 and EC50 values after 1 h incubation with cis-[Pt(bapda)Cl2] and cisplatin (cis-[PtCl2(NH3)2]) and subsequent incubation with drug-free medium in human ovarian carcinoma cell lines A2780 and A2780R (pEC50,mean ± SD, n = 4).
Short-time exposure
compound A2780 A2780R RF
cis-[Pt(bapda)Cl2] pEC50 EC50, mM
1.57±0.06 26.9 10-3
1.24±0.13
57.5 10-3 2.1 cisplatin
pEC50
EC50, mM
1.69±0.26 20.0 10-3
0.45±0.35
>100 nd
RF (resistance factor) = EC50 (A2780R)/ EC50 (A2780), nd = not determinable
Decreased biological activity of cis-[Pt(bapda)Cl2] compared to that of long-time exposure (described in chapter 6, in Table 6.1) is observed. This observation suggests that the cells are able to recover from the damage caused by cis-[Pt(bapda)Cl2] during the post-incubation time with drug-free medium. Similar behavior was observed for cisplatin, with higher EC50 values in short-time exposure experiments. Nevertheless, a higher cytotoxic activity of cis-[Pt(bapda)Cl2] after 1 h exposure is observed, compared to that of cisplatin. Additionally, cis-[Pt(bapda)Cl2] forms larger amounts of DNA-adducts, (described in chapter 6, in Fig.6.11) compared to those of cisplatin, in both cell lines under the same conditions. Therefore, it is not surprising that the cytotoxic activity of cis-[Pt(bapda)Cl2] after 1 h exposure is indeed higher than that of cisplatin.
Significant differences between the cytotoxic activity of cis-[Pt(bapda)Cl2] in the A2780 and the A2780R cells have been found (p = 0.038); therefore, cross-resistance to platinum in A2780R is observed also after short-time exposure, and it is in agreement
with a resistance factor RF > 2. It is worthy to note that the DNA platination of cis-[Pt(bapda)Cl2] found in the A2780R cells is significantly higher that those in the sensitive counterpart (see Chapter 6, Fig. 6.11), which is not in agreement with the cross-resistance in the A2780R cells. Higher levels of DNA platination in the A2780R cells accompanied by cross-resistance to platinum strongly suggests that the cells are able to tolerate the DNA damage caused by cis-[Pt(bapda)Cl2].
7.3.4. Effect of bafilomycin A1 and monensin on the cytotoxicity activity
Cytotoxic activity of cis-[Pt(bapda)Cl2] in the A2780 and the A2780R cells preincubated with bafilomycin A1, or monensin has been investigated and compared to that of cis-[Pt(bapda)Cl2] in the absence of inhibitors (performed in parallel cultures).
The results are summarized in Fig. 7.11 and 7.12. Non-treated cells were used as control to calculate the percentage of viable cells in each well after the incubation with 10 M of cis-[Pt(bapda)Cl2]. In addition, bafilomycin A1- and monensin-treated cells in the absence of cis-[Pt(bapda)Cl2] were used to control the cytotoxicity of the inhibitors in the cells.
0 15 30 45
0 15 30 45
A2780R cis-[Pt(bapda)Cl
2]
% viable cells
bafilomycin A1 no bafilomycin A1
A2780 cis-[Pt(bapda)Cl
2]
Fig. 7.11. Effect of bafilomycin A1 (100 nM, 1 h) on the cytotoxic activity of cis-[Pt(bapda)Cl2] in the A2780 and the A2780R cells, compared to that of cis-[Pt(bapda)Cl2] without bafilomycin A1 preincubation.
(mean ± SD, n = 4).
The concentrations of bafilomycin A1 (100 nM) and monensin (20 M) did not alter cell viability (> 90% viable cells) in either the A2780 or the A2780R cells.
Preincubating the A2780R cells with bafilomycin A1 for 1 h did not alter the cytotoxic activity of cis-[Pt(bapda)Cl2]. In contrast, a small but statistically significant decrease of the activity in the A2780 cells (p = 0.0485) has been observed when the A2780 cells are preincubated with bafilomycin A1. In contrast, preincubating the A2780 and the A2780R cells with monensin for 1 h shows a significant effect in the activity of cis-[Pt(bapda)Cl2].
The cytotoxic activity in A2780 cells is not dramatically altered (p = 0.0443); however, the activity of cis-[Pt(bapda)Cl2] in the A2780R cells is largely increased (p = 0.0001) when the cells are pretreated with monensin (Fig. 7.12).
0 15 30 45
0 15 30 45
A2780R cis-[Pt(bapda)Cl
2]
% viable cells
monensin no monensin
A2780 cis-[Pt(bapda)Cl
2]
Fig. 7.12. Effect of monensin (20 M, 1 h) on the cytotoxic activity of cis-[Pt(bapda)Cl2] in the A2780 and the A2780R cells, compared to that of cis-[Pt(bapda)Cl2] without monensin treatment. (mean ± SD, n = 4).
As shown in the control studies with acridine orange, bafilomycin A1 alters the vesicular pH in both cell lines. In addition, bafilomycin A1 has been shown to influence the cellular processing of the platinum(II) compound (Fig. 7.7 and 7.6). Nevertheless, bafilomycin A1 does not induce large alterations on the cytotoxic activity of cis-[Pt(bapda)Cl2]. This observation suggests that the lysosomal sequestration of the platinum(II) compound does not play an important role for the cytotoxic activity.
Interestingly, the cytotoxic activity of cis-[Pt(bapda)Cl2] in monensin-preincubated A2780R cells was found to be increased 1.7-fold, compared to the monensin-free cells. Studies with acridine orange have shown that monensin does not
alter the vesicular pH in either the A2780 or the A2780R cells. Therefore, the cross-resistance found for this platinum(II) compound might not be necessarily related to the lysosomal sequestration. Nevertheless, preincubating the cells with monensin shows alteration in the cellular processing of cis-[Pt(bapda)Cl2], where the compound is accumulated in the acidic lysosomes, as well as being more visible in the cytoplasm with the appearance of new vesicles (Fig. 7.5 and 7.6).
7.3.5. Intracellular platinum accumulation in human ovarian carcinoma cells Intracellular accumulation of cis-[Pt(bapda)Cl2] compared to that of cisplatin after 1 h exposure has been investigated in the A2780 and the A2780R cells (Fig. 7.13).
0 5 10 50 100
0 5 10 50 100
cis-[Pt(bapda)Cl
2]
nmol Pt/million cells
A2780 A2780R
cisplatin
Fig. 7.13. Intracellular platinum concentration in the A2780 and the A2780R cells after incubation for 1 h with 50 M final concentration of cisplatin (cis-[Pt(NH3)2Cl2] and cis-[Pt(bapda)Cl2] (mean ± SD, n = 5-6).
Lipophilic carrier ligands have been used to synthesize new platinum compounds which can be taken up more efficiently within the cells, i.e. oxaliplatin.24 Therefore, it is not surprising that the cellular accumulation of cis-[Pt(bapda)Cl2] is significantly higher than that of cisplatin in both the A2780 and the A2780R cells. More interestingly, the intracellular accumulation of cis-[Pt(bapda)Cl2] in the A2780R cells is significantly higher than that in the A2780 cells (p = 0.004). This accumulation is not in agreement with the observed cross-resistance in the A2780R cells. This observation suggests that the resistance to platinum of cis-[Pt(bapda)Cl2] is not related to the ability of cis-[Pt(bapda)Cl2] to be taken up by the cells, since a decreased intracellular accumulation in the A2780R would be expected, compared to that in the A2780 cells.
Nevertheless, higher accumulation of cis-[Pt(bapda)Cl2] in the A2780R cells correlates with the high levels of DNA-platination found for this compound.
Additionally, a lower accumulation of cisplatin in the A2780R cells, compared to the sensitive counterpart, has been observed. This is in agreement with reported results, where it was found that one of the most evident resistant mechanisms against cisplatin is a decreased influx.25
7.3.6. Bafilomycin A1 and monensin effect on the intracellular accumulation To investigate the influence of bafilomycin A1 in the intracellular accumulation of cis-[Pt(bapda)Cl2] in the A2780 and the A2780R cells, preincubation with bafilomycin A1 was performed, and compared to those of normal accumulation of cis-[Pt(bapda)Cl2] (performed in parallel cultures). The results are summarized in Fig. 7.14.
0 30 60 90
0 30 60 90
A2780R cis-[Pt(bapda)Cl
2]
ng Pt/million cells
no BafA
1
BafA1
A2780 cis-[Pt(bapda)Cl
2]
Fig. 7.14. Influence of bafilomycin A1 (100 mM) on the intracellular platinum concentration of cis-[Pt(bapda)Cl2] after 1 h incubation with 50 M of cis-[Pt(bapda)Cl2] in A2780 and A2780R cells, preincubated or non-preincubated with bafilomycin A1 (100 nM) (mean ± SD, n = 5).
Bafilomycin A1 does not influence the intracellular accumulation of cis-[Pt(bapda)Cl2] in either the A2780 or the A2780R cells (p>0.05). The effect of the alteration in the lysosomal/endosomal pH by treatment with bafilomycin A1 on the cellular uptake of 14C-carboplatin in cisplatin-sensitive and resistant human adenocarcinoma cell lines has been recently reported.1 A fourfold decrease in the accumulation of the 14C-carboplatin after treatment with the inhibitor in the sensitive cell lines was obtained. It was concluded that a defective endosomal/lysosomal acidification
may be partly responsible for the cisplatin-resistance due to a reduced uptake, resulting in less drug-DNA interaction. Additionally, accumulation studies of macromolecular platinum drugs in human ovarian carcinoma cisplatin-sensitive and cisplatin-resistant cell lines treated with bafilomycin A1 have been recently reported.26 In the presence of bafilomycin A1 a decreased intracellular accumulation of these compounds was observed, suggesting endocytosis as an uptake pathway. Therefore, the lack of effect of bafilomycin A1 in the accumulation process of cis-[Pt(bapda)Cl2] in the A2780 or the A2780R cells suggests that the accumulation of this new platinum(II) compound is not influenced by the lysosomal/endosomal pH, and that this compound might not be taken up via endocytosis. Nevertheless, endocytosis is a complex cellular process, where multiple methods of internalization are present.27 These include clathrin-dependent endocytosis, clathrin-independent endocytosis, macropinocytosis and internalization via caveolae.27
Preincubation of both cell lines with monensin in the intracellular platinum accumulation studies of cis-[Pt(bapda)Cl2] was also performed, and the results are summarized in Fig. 7.15. The intracellular accumulation of cis-[Pt(bapda)Cl2] in monensin-free cells was performed in parallel cultures to allow comparison.
0 20 40 60 80 100 120 140
0 20 40 60 80 100 120 140
A2780R cis-[Pt(bapda)Cl
2]
ng Pt/million cells
no monensin monensin
A2780 cis-[Pt(bapda)Cl2]
Fig. 7.15. Influence of monensin (20 M) on the intracellular platinum concentration of cis-[Pt(bapda)Cl2] after 1 h incubation with 50 M of cis-[Pt(bapda)Cl2] in A2780 and A2780R cells (mean ± SD, n = 3-4).
Surprisingly, monensin has a significant influence (p < 0.05) on the intracellular accumulation of cis-[Pt(bapda)Cl2] in the A2780R cells, where decreased levels of platinum in monensin-treated cells compared to monensin-free cells are observed.
Moreover, no effect of the monensin is observed in the intracellular accumulation of cis-[Pt(bapda)Cl2] in A2780 cells (p > 0.05). The alteration in the cellular accumulation of cis-[Pt(bapda)Cl2] in the A2780R cells is not in agreement with the increased cytotoxicity of the platinum(II) compound in these cells. Nevertheless, inhibition studies towards cytotoxic activity of cis-[Pt(bapda)Cl2] were performed using 24 h incubation of the platinum(II) compound, and during this long-time exposure the effect of monensin might be reversible.
It appears that the monensin effect towards platinum compounds accumulation within cancer cells has not been reported. However, studies where monensin preincubation caused a significant decrease in daunorubicin accumulation in cytoplasts have been reported.16 Monensin blocks intracellular transport of protein at the level of the Golgi apparatus, with no known effect on the protein synthesis. The effect of this inhibitor is therefore considered to be on transport rather than on processing.29 More interestingly, monensin prevents endosomal acidification and maturation, and it has an important effect on the Golgi apparatus.29 Swelling response of the Golgi apparatus after monensin treatment has been reported in many cases.29 Monensin causes swelling of Golgi apparatus through a Na+-in, H+-out exchange across the membranes leading to a net uptake of Na+ and Cl¯ and entry of water. In endocytosis, monensin does not prevent internalization, but prevents degradation of internalized molecules. Additionally, endocytosis involves both acidic and non-acidic compartments, and monensin inhibits those processes that normally occur in acidic compartments. Thus, monensin is an accepted inhibitor to study the Golgi apparatus function, and to localize and identify the molecular pathways of subcellular vesicular traffic involving acid compartments such as lysosomes or endosomes. In particular, cis-[Pt(bapda)Cl2] is accumulated in acidic lysosomes and the treatment with monensin shows small alteration in the cellular processing of this compound. Accumulation of cis-[Pt(bapda)Cl2] in other vesicles, beside the acidic lysosomes in monensin-treated A2780R cells has been observed.
Alterations in the Golgi apparatus in the A2780R cells due to monensin preincubation
may be involved in the cellular processing of cis-[Pt(bapda)Cl2]; however, not disruption of Golgi apparatus have been observed by fluorescence microscopy using the specific Bodipy Ceramide TR dye (images not shown).
7.4. Concluding remarks and outlook
To investigate the role of the lysosomal accumulation of cis-[Pt(bapda)Cl2] in both cell lines, as a possible uptake pathway, two inhibitors have been used. Incubation with bafilomycin A1 has shown to alter the intravesicular pH of the A2780 and the A2780R cells to a large extent, as confirmed by the non-specific localization of acridine orange in both the A2780 and the A2780R cells. The studies performed using bafilomycin A1 in both cell lines, previous incubation with cis-[Pt(bapda)Cl2], has shown neither to effect the biological activity nor to influence the intracellular accumulation of this platinum(II) compound. In contrast, cellular processing studies of cis-[Pt(bapda)Cl2] using fluorescence microscopy show that the alterations in the pH gradient within the cells using bafilomycin A1 have an effect on the cellular distribution of the platinum(II) compound in both cell lines. The involvement of defective vacuolar acidification in cisplatin resistant cells has been previously reported.1 These studies suggested that defective endosomal/lysosomal acidification may, at least partly be responsible for cisplatin resistance due to its reduced uptake, with less drug reaching the cytotoxic target.1 It has been reported that altered intracellular drug distribution in resistant cell lines is often closely related to alterations in drug accumulation, making it difficult to discriminate between both processes.15 In the case of cis-[Pt(bapda)Cl2], a different cellular distribution seems not to be related neither to its biological activity nor to its cellular accumulation in both cell lines.
The same studies using monensin displays a significant effect in both the biological activity and the intracellular accumulation of cis-[Pt(bapda)Cl2] in the cisplatin-resistant cell line (A2780R). Nevertheless, according to the labeling with acridine orange, monensin shows no alterations in the intracellular pH gradient in the A2780 and the A2780R cells. Perturbation of the pH by proton ionophores, like monensin, raise the pH of acidified compartments, and affects protein transport through the biosynthetic and endocytotic pathway.22, 23, 28 Preincubation of the A2780 and the A2780R cells with monensin does not seem to affect the intravesicular pH, and
reversibility of the monensin effect has been also dismissed, because keeping the inhibitor in the cell medium, together with acridine orange, has also no effect on the intravesicular pH. However, it is important to note that the reversibility of monensin has been observed in many cases a few hours after its use.29 Therefore, long-time experiments, i.e. cytotoxicity or cellular processing 24 h after monensin incubation, have to be carefully analyzed, because the monensin effect meanwhile might have been reversed. Nevertheless, it can be suggested that the significant increase in the cytotoxic activity of cis-[Pt(bapda)Cl2] in the A2780R cells, and the decreased intracellular accumulation of this compound in the cisplatin-resistant cells after monensin preincubation, might not be related to pH alterations. This last observation is in agreement with the results obtained using bafilomycin A1.
It has been suggested that the vesicular sequestration is a saturable process that strongly depends on the cytoplasmic concentration.16 A diffuse cytoplasmic/nuclear drug distribution, when the capacity of the vesicular system is exceeded, has been observed.16 For this reason, the altered intracellular drug distribution in resistant cells is partly a result of the activity of an efflux pump that brings the levels of intracellular drug down to a concentration that falls within the capacity of the cytoplasmic organelles to sequester the drug.30 This observation can explain the decrease drug uptake of cis-[Pt(bapda)Cl2] observed in monensin-treated A2780R cells, where more compound is localized in the cytoplasm and in different vesicles of the cells after monensin preincubation. More drug is distributed over the cells; therefore, the cells increase the efflux to balance the levels of compound between cytoplasm and acidic compartments. Nevertheless, this is not in agreement with the increased cytotoxicity displayed by cis-[Pt(bapda)Cl2] in the monensin-treated A2780R cells, since lower accumulation of the platinum(II) compound inside the cell should lead to a lower cytotoxic activity. A possible explanation for the increase of the cytotoxicity of cis-[Pt(bapda)Cl2] in monensin-preincubated A2780R cells might be related to the increased permeability in the intracellular membranes due to the monensin preincubation. Larger amounts of cis-[Pt(bapda)Cl2] may be able to reach the biological target in monensin-treated cells, since the monensin is able to alter the permeability of the internal membranes.
References
1. Chauhan, S. S.; Liang, X. J.; Su, A. W.; Pai-Panandiker, A.; Shen, D. W.; Hanover, J. A.;
Gottesman, M. M., Br. J. Cancer 2003, 88, 1327-1334.
2. Kalayda, G. V.; Jansen, B. A. J.; Wielaard, P.; Tanke, H. J.; Reedijk, J., J. Biol. Inorg. Chem.
2005, 10, 305-315.
3. Kalayda, G. V.; Wagner, C. H.; Buss, I.; Reedijk, J.; Jaehde, U., BMC Cancer 2008, 8.
4. Millot, C.; Millot, J. M.; Morjani, H.; Desplaces, A.; Manfait, M., J. Histochem. Cytochem. 1997, 45, 1255-1264.
5. Safaei, R.; Larson, B. J.; Cheng, T. C.; Gibson, M. A.; Otani, S.; Naerdemann, W.; Howell, S. B., Mol. Cancer Ther. 2005, 4, 1595-1604.
6. Beck, W. T., Biochem. Pharmacol. 1987, 36, 2879-2887.
7. Katano, K.; Safaei, R.; Samimi, G.; Holzer, A.; Tomioka, M.; Goodman, M.; Howell, S. B., Clin.
Cancer Res. 2004, 10, 4578-4588.
8. Safaei, R.; Katano, K.; Larson, B. J.; Samimi, G.; Holzer, A. K.; Naerdemann, W.; Tomioka, M.;
Goodman, M.; Howell, S. B., Clin. Cancer Res. 2005, 11, 756-767.
9. Kroemer, G.; Jaattela, M., Nat. Rev. Cancer 2005, 5, 886-897.
10. Ouar, Z.; Bens, M.; Vignes, C.; Paulais, M.; Pringel, C.; Fleury, J.; Cluzeaud, F.; Lacave, R.;
Vandewalle, A., Biochem. J. 2003, 370, 185-193.
11. Warren, L.; Jardillier, J. C.; Ordentlich, P., Cancer Res. 1991, 51, 1996-2001.
12. Klohs, W. D.; Steinkampf, R. W., Mol. Pharmacol. 1988, 34, 180-185.
13. Schindler, M.; Grabski, S.; Hoff, E.; Simon, S. M., Biochemistry 1996, 35, 2811-2817.
14. Marquardt, D.; Center, M. S., J. Natl. Cancer Inst. 1991, 83, 1098-1102.
15. Larsen, A. K.; Escargueil, A. E.; Skladanowski, A., Pharmacol. Ther. 2000, 85, 217-229.
16. Slapak, C. A.; Lecerf, J. M.; Daniel, J. C.; Levy, S. B., J. Biol. Chem. 1992, 267, 10638-10644.
17. Sehested, M.; Skovsgaard, T.; Roed, H., Biochem. Pharmacol. 1988, 37, 3305-3310.
18. Altan, N.; Chen, Y.; Schindler, M.; Simon, S. M., J. Exp. Med. 1998, 187, 1583-1598.
19. Drose, S.; Altendorf, K., J. Exp. Biol. 1997, 200, 1-8.
20. Barasch, J.; Gershon, M. D.; Nunez, E. A.; Tamir, H.; Alawqati, Q., J. Cell Biol. 1988, 107, 2137- 2147.
21. Liang, X. J.; Shen, D. W.; Garfield, S.; Gottesman, M. M., Cancer Res. 2003, 63, 5909-5916.
22. Maxfield, F. R., J. Cell Biol. 1982, 95, 676-681.
23. Tartakoff, A. M., Cell 1983, 32, 1026-1028.
24. Di Francesco, A. M.; Ruggiero, A.; Riccardi, R., Cell. Mol. Life Sci. 2002, 59, 1914-1927.
25. Siddik, Z. H., Oncogene 2003, 22, 7265-7279.
26. Garmann, D.; Warnecke, A.; Kalayda, G. V.; Kratz, F.; Jaehde, U., J. Control. Release 2008, 131, 100-106.
27. Watson, P.; Jones, A. T.; Stephens, D. J., Adv. Drug Deliv. Rev. 2005, 57, 43-61.
28. Tartakoff, A.; Vassalli, P., J. Cell Biol. 1978, 79, 694-707.
29. Mollenhauer, H. H.; Morre, D. J.; Rowe, L. D., Biochim Biophys Acta 1990, 1031, 225-246.
30. Come, M. G.; Bettaieb, A.; Skladanowski, A.; Larsen, A. K.; Laurent, G., Int. J. Cancer 1999, 81, 580-587.
