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
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6 6 . .
A A n ne ew w f fl lu uo or r e e sc s ce en nt t p pl la at ti in nu um m( (I II I) ) c c om o mp po ou un nd d c co on nt ta ai in ni in ng g N N , , N N ’ ’ - - b b i i s s ( ( a a n n t t h h r r a a c c e e n n - - 9 9 - - y y l l m m e e t t h h y y l l ) ) p p r r o o p p a a n n e e - - 1 1 , , 3 3 - - d d i i a a m m i i n n e e (b ( ba ap pd da a) ) a a s s a a c c a a r r r r ie i er r l li ig ga an nd d: : I In n vi v it tr ro o c cy yt to ot to ox xi ic ci it ty y a an nd d c ce el ll lu ul la ar r
pr p ro oc ce es ss si in ng g i in n t th he e A A 27 2 78 80 0 o ov va ar ri ia an n c ca ar rc ci in no om ma a c ce el ll l l li in ne e .* . *
Abstract
The cytotoxic activity of cis-[Pt(bapda)Cl2] and the platinum free-molecule (bapda) against a pair of human ovarian carcinoma cell lines, namely the cisplatin- sensitive A2780 and its resistant counterpart A2780R cells, has been investigated and compared to the biological activity of cisplatin. Cross resistance to platinum has been found after 48 h incubation with cis-[Pt(bapda)Cl2] in the A2780R cells. Basic mechanistic studies towards the understanding of the mode of action of this new platinum compound have been undertaken, such as inactivation by the GSH molecule in the A2780R cells. The cellular processing of this platinum(II) compound, as compared to its corresponding free ligand in the same cells and under same conditions, has also been investigated by fluorescence microscopy. Different cellular processing of bapda and cis-[Pt(bapda)Cl2] has been observed when GSH levels were depleted in the A2780R cells.
* Parts of this chapter are based on:
Patricia Marqués-Gallego, Hans den Dulk, Jaap Brouwer, Stefania Tanase, Ilpo Mutikainen, Urho Turpeinen, Jan Reedijk, Biochemical Pharmacology, 2009, 78, 365-373
6.1. Introduction
In the last three decades many efforts have been dedicated to establish the factors related to the antitumor activity of different platinum complexes. Even though the molecular mechanistic studies of cisplatin and analogues have been thoroughly studied,1, 2 the cellular response to these compounds is still poorly understood. Much of the current understanding of the mechanism of action of platinum-based drugs comes from studies with cisplatin. An interesting approach based on a fluorescein-labeled cisplatin derivative3 has provided new insights in the cellular response to cisplatin, using fluorescence microscopy.4, 5 On the other hand, the use of fluorescent carrier ligands, that can easily coordinate to the platinum(II) ion, is another successful approach in the search for new fluorescent platinum-based drugs. Thus, studies concerning antitumor drugs and cellular response open new options towards the design and development of new platinum compounds.6-11 Furthermore, the resistance of certain tumors to cisplatin treatment is one of the major drawbacks of the drug in the clinic, and new platinum compounds that overcome this resistance are still highly desired. Chapter 5 deals with the synthesis of a new fluorescent platinum(II) compound. The use of bapda as carrier ligand addresses three of the well-known cisplatin resistance mechanisms,12 namely (i) decreased drug uptake, (ii) inactivation by thiol-containing molecules, and (iii) increased DNA repair. A more hydrophobic carrier ligand, such as bapda, can be used to increase the accumulation of the platinum compound within the cell, since a correlation between the carrier ligand lypophilicity and drug accumulation has been found.13 In addition, the steric hindrance around the platinum atom in of cis-[Pt(bapda)Cl2] might decrease the possible interaction with thiol-containing molecules, such as GSH or MT, which are abundantly present within the cells and which are known to inactivate platinum compounds as part of a resistance mechanism.14 In addition to this, the use of carrier ligands containing an intercalator may increase the interaction with the DNA molecules, raising in this way the biological damage with impact on the repair process.15
In Chapter 5, the synthesis, characterization and solution behavior of cis-[Pt(bapda)Cl2] has been described. This chapter deals with the biological activity of cis-[Pt(bapda)Cl2] against the A2780 and the A2780R cells, as compared to its corresponding carrier ligand (bapda) and cisplatin.
6.2. Experimental Section
6.2.1. Cell lines, culture conditions and cytotoxicity assays
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).
6.2.2. Digital fluorescence microscopy
Fluorescence digital imaging of living cells was performed with the pair of human ovarian carcinoma the A2780 and the A2780R cell lines. For the living cell observations the cells were grown in 35 mm culture dish with a cover slip incorporated in the bottom to 30-50% confluence in Dulbecco´s modified Eagle´s Medium (DMEM) (Gibco BRL
TM, Invitrogen Corporation, The Netherlands) supplemented with 10% heat-inactivated fetal calf serum (Hyclone, Perbio Science, The Netherlands), penicillinG Sodium (100 units/ml: Dufecha, Biochemie BV, The Netherlands), streptomycin (100 μg/ml: Dufecha, Biochemie BV, The Netherlands) and GlutaMAXTM 100x (Gibco BRL TM, The Netherlands). Before the incubation with the compounds, the cells were washed twice with PBS. Subsequently, the compounds were added to the cells at a final concentration of 10 M in serum-free and phenol red-free medium for 15 min at 37 °C and 7% CO2
atmosphere. After incubation, the cells were washed twice with PBS, and drug-free medium supplemented with 10% heat-inactivated fetal calf serum, penicillinG Sodium (100 units/ml), streptomycin (100 μg/ml) and GlutaMAXTM 100x, was added to the cells.
Phase contrast images and the corresponding fluorescence images were taken at different time points after incubation at 37 °C and 5.2% CO2 atmosphere. After imaging the medium was refreshed, and the cells were kept at 37 °C in a 7% CO2 incubator, to study the accumulation twenty-four hours after incubation with the ligand bapda or cis-[Pt(bapda)Cl2].
Pictures were taken using a fluorescence 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. To detect the fluorescence signal of bapda ligand and cis-[Pt(bapda)Cl2] a filter for ex 377 nm (time exposure = 10 ms) was used. Digital images were taken with a cooled CCD camera (F-View, Olympus The Netherlands). Images were processed by using Cell M software.
LysoTrackerTM Red DND-99 (Molecular Probes, Leiden, The Netherlands) was used to stain the vesicles observed in the cytosol. The staining was performed 24 h after incubation with the corresponding compound. LysoTrackerTM Red DND-99 was added to the culture medium in a final concentration of 50 nM. After 15 min of incubation with the dye, the cells were washed twice with PBS, and complete phenol red-free medium was added before imaging. For detecting fluorescence of LysoTrackerTM localized in lysosomes TRITC-filter (ex 573 nm; time exposure = 10 ms) was used.
6.2.3. Reaction of cis-[Pt(bapda)Cl2] with 9-Ethylguanine
The cis-[Pt(bapda)Cl2] (2.44 mg, 3.4 mmol) was allowed to react with 9-ethylguanine (9-EtG, 1.2 mg, 6.7 mmol) in DMF-d7 at 37 ºC to investigate how this compound interact with nucleobases. Cis-[Pt(bapda)Cl2] and 9-EtG were both dissolved in DMF-d7 and 1H NMR spectra were recorded at 37 ºC and followed over time. After 24 h of reaction the 195Pt NMR spectrum at 37 ºC was recorded.
6.2.4.Measurement of DNA platination
The DNA platination displayed by cis-[Pt(bapda)Cl2] in both the A2780 and in the A2780R cells was performed as described in Chapter 3 (section 3.2.5), and compared to the DNA platination of cisplatin.
6.2.5. DNA fluorescence titration
Fluorescence titration of cis-[Pt(bapda)Cl2] was performed as described previously.11 Small aliquots of a concentrated calf thymus DNA solution were added to 50 M solutions of cis-[Pt(bapda)Cl2] ranged from 0:1 to 4:1 base pairs:cis-[Pt(bapda)Cl2]. Fresh solutions of cis-[Pt(bapda)Cl2] in DMF diluted with phosphate buffer were prepare fresh and the titration spectra were recorded after mixing.
6.2.6. Statistical analysis
In the biological studies such as DNA platination and cytotoxic activity, the Student’s t-test was performed for statistical comparison. A p-value < 0.05 was considered as statistically significant.
6.3. Results and discussion
6.3.1. Cytotoxicity assay in human ovarian carcinoma cell lines
The cytotoxic activity of cis-[Pt(bapda)Cl2], the free ligand bapda, and cisplatin as reference compound against the A2780 and the A2780R cell lines was studied. They were compared with regard to their cytotoxic activity against the same human tumor cell lines under the same conditions, in order to determine to which extent cis-[Pt(bapda)Cl2] improves the cytotoxic behavior of its corresponding platinum-free molecule bapda, a potential intercalator. The EC50 (mM) values (EC50 is the drug concentration that produces 50% of the maximum possible response) after 48 h incubation are listed in Table 6.1.
Cis-[Pt(bapda)Cl2] shows a high cytotoxic activity against the human ovarian carcinoma sensitive and cisplatin-resistant cell lines. Moreover, significant differences (p < 0.05) between pEC50 values for cis-[Pt(bapda)Cl2] and its free carrier ligand bapda against the A2780 cell line were found, indicating that the coordination compound is significantly more active against the cisplatin-sensitive human ovarian carcinoma A2780 cell line than its free carrier ligand. In addition, cis-[Pt(bapda)Cl2] displays the same level of cytotoxic activity as cisplatin, with a pEC50 value not significantly different (p > 0.05) from than found for cisplatin in the same A2780 cell line. However, the platinum-free molecule bapda shows significantly lower activity against the A2780 cell line when compared to cisplatin.
On the other hand, when cisplatin and cis-[Pt(bapda)Cl2] are compared regarding their cytotoxic activity against the A2780R cell line, significant differences (p < 0.001) can be observed. The same was found when the cytotoxic activity of bapda was compared to cisplatin cytotoxicity in the A2780R cell line, where significant differences
were observed (p < 0.0001). Thus, both bapda and cis-[Pt(bapda)Cl2], have improved the cytotoxic activity against the A2780R compared to cisplatin.
In addition, it is worth to note that cis-[Pt(bapda)Cl2] shows significant differences (p < 0.001) in the cytotoxic activity between the A2780 and the A2780R cells, which suggests cross-resistance of cis-[Pt(bapda)Cl2] in the human ovarian carcinoma cisplatin-resistant cell line A2780R; this cross-resistance is indicated also by the resistance factor (RF > 2).
Table 6.1. pEC50 and EC50 values after 48 h incubation with cis-[Pt(bapda)Cl2], ligand bapda, and cisplatin as a reference compound in human ovarian carcinoma cell lines theA2780 and the A2780R (pEC50,mean ± SD, n = 4 - 5).
compound A2780 A2780R RF* A2780R-L-BSO
bapda pEC50 EC50 (mM)
2.46±0.03 3.5 x 10-3
2.40±0.05
4.0 x 10-3 1.1
2.17±0.09 6.8 x 10-3 cis-[Pt(bapda)Cl2]
pEC50
EC50 (mM)
2.64±0.13 2.3 x 10-3
2.15±0.06
7.1 x 10-3 3.1
2.19±0.07 6.5 x 10-3 cisplatin
pEC50
EC50 (mM)
2.64±0.13 2.3 x 10-3
1.70±0.05
20.0 x 10-3 8.7
2.12±0.06 7.6 x 10-3
*RF = EC50 (A2780R)/ EC50 (A2780)
A2780R-L-BSO = A2780R pre- and co-incubated with L-BSO
As reported in the literature, glutathione (GSH) is a sulfur-containing molecule abundantly present in many cells, and elevated levels of GSH mediate cellular resistance against platinum cancer drugs, such as cisplatin.17 Divalent platinum is highly reactive towards sulfur of glutathione and may provide an explanation for cross-resistance of cis-[Pt(bapda)Cl2] with cisplatin in the A2780R cells. Depletion of GSH levels by L-buthionine-S,R-sulfoximine (L-BSO) in the A2780R cells was used to further investigate this possibility (Table 6.1). According to the cytotoxic activity obtained in GSH-depleted A2780R cells, no significant differences were found (p >0.05) when compared to untreated A2780R cells. This observation suggests that the cross-resistance with cisplatin found for cis-[Pt(bapda)Cl2] after 48 h stems from a different resistance mechanism than inactivation by thiol-containing molecules. It has been proposed that bulkier ligands around the platinum(II) ion might prevent or reduce the approach of
glutathione (GSH). The reactivity of cis-[Pt(bapda)Cl2] towards GSH has been studied by
195Pt NMR, and the results are discussed below.
6.3.2. Interaction of cis-[Pt(bapda)Cl2] with GSH
In order to establish the possible reactivity of cis-[Pt(bapda)Cl2] towards glutathione, cis-[Pt(bapda)Cl2] and GSH were allowed to react in a 1:4 molar ratio in DMF/PBS at 37 °C, and the reaction was monitored every 4 h by 195Pt NMR spectroscopy for 24 h. Intracellular GSH concentrations are known to be in the range of 10-100 nM;18 therefore, the platinum(II) compound was reacted with an excess of GSH in order to compare to the resistant cell line. No interaction between cis-[Pt(bapda)Cl2] and the GSH was detected, as a constant single peak at -2182 ppm, corresponding to unreacted cis-[Pt(bapda)Cl2] was detected up to 24 h (Fig. 6.1). This suggests that cis-[Pt(bapda)Cl2] is not susceptible to GSH deactivation in the A2780R cell line, as has been observed when depleted GSH levels in the A2780R cells were studied for cytotoxic activity (Table 6.1).
Fig. 6.1. 195Pt NMR spectrum (300 MHz) of cis-[Pt(bapda)Cl2)] in DMF/PBS at 37 °C reacted with 4 eq.
GSH over time (spectra from bottom to top: after 4 h, 8 h, and 24 h)
6.3.3. Digital fluorescence microscopy
Cellular processing of the ligand bapda, and cis-[Pt(bapda)Cl2] has been investigated using fluorescence microscopy. The cells were incubated in parallel culture dishes with the ligand bapda and with cis-[Pt(bapda)Cl2] for 15 min at 10 M final concentration in serum-free medium. After removal of the platinum compound, its cellular processing in the A2780 and the A2780R cells was compared. In addition, the
comparison between the platinum-free molecule and its corresponding platinum(II) compound was carried out.
Incubation with both compounds, the ligand bapda and cis-[Pt(bapda)Cl2], has been chosen relatively short due to the high cytotoxic activity against the A2780 and the A2780R cells; nevertheless, both compounds were able to enter the cells within 15 min of incubation. After 24 h the compounds are still visible inside the cells, with retention within large vesicles (Fig. 6.3 and 6.4).
According to the cellular processing images of cis-[Pt(bapda)Cl2] shown in Fig.
6.2, the distribution of the platinum(II) compound over time in the A2780 and the A2780R is slightly different. Quickly after incubation with cis-[Pt(bapda)Cl2], accumulation in the human ovarian carcinoma cisplatin-resistance cell line in vesicles is observed. This accumulation remains constant over time up to 24 h after incubation (Fig. 6.3). In contrast, the cellular distribution of cis-[Pt(bapda)Cl2] within the A2780 cell line changes over time (Fig. 6.2). After incubation with the compound the fluorescence signal is visible in some vesicles close to the cell nucleus. However, three hours after the incubation, cis-[Pt(bapda)Cl2] is accumulated in vesicles extended over the cytosol. This accumulation remains visible 24 h after the incubation (Fig. 6.4). In both cell lines the vesicles were identified as acidic lysosomes (Fig. 6.3 and 6.4) with the use of a specific stain (LysoTrackerTM Red DND-99). In addition, as it is clear from the phase-contrast images, the number of vesicles around the nucleus present in both cell lines increases over time. This observation might be associated to intracellular transport mechanisms in the cells.
Dinuclear platinum(II) compounds trapped in lysosomal vesicles in human ovarian carcinoma (A2780R) cells and in the human osteosarcoma (U2OS) cell line have been reported earlier.6 The reported accumulation in the A2780R was found constant over time, which was not observed for the sensitive A2780 cell line.6 This observation suggested vesicular sequestration of the dinuclear complexes, which might be due to a specific resistance mechanism present in the A2780R cell line. In the same studies, after 24 h of incubation with the free ligand no vesicles containing the compounds were observed, suggesting that the sequestration in acidic vesicles in the A2780R cell line is a specific response to the corresponding platinum compounds.6
A
B
C
D
E
F
Fig. 6.2. Cellular distribution of cis-[Pt(bapda)Cl2] in the A2780 human ovarian carcinoma cell line (A, B and C) and cellular processing of cis-[Pt(bapda)Cl2] in the A2780R human ovarian resistant cell line (D,E and F) Images A and D: 10 min after incubation. Images B and E: 1 h after incubation. Images C and F: 3 h after incubation. Phase-contrast images are shown on the left side, corresponding fluorescent images are in the middle, and superimposed images are on the right side.
A B
D C
Fig. 6.3. Cellular distribution of cis-[Pt(bapda)Cl2] in the A2780R human ovarian carcinoma cisplatin- resistant cell line 24 h after incubation, 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).
A
C
B
D
Fig. 6.4. Cellular distribution of cis-[Pt(bapda)Cl2] in the A2780 the human ovarian carcinoma cell line 24 h after incubation, 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).
However, in the cellular processing of cis-[Pt(bapda)Cl2] such a specificity was not found, since a similar accumulation of the free ligand bapda and cis-[Pt(bapda)Cl2] was observed 24 h after incubation in acidic vesicles in the A2780R cells. Moreover, as mentioned above, the same lysosomal sequestration is observed in the sensitive human ovarian carcinoma cell line A2780 treated with cis-[Pt(bapda)Cl2] (Fig. 6.4), which suggests that such accumulation is not related to the resistance mechanisms. Organic drugs, such as daunorubicin and mitoxantrone were found to be accumulated in acidic vesicles,19 which indicates that this might be a protective response of the cells to different external agents. In addition, the cellular distribution of the free ligand bapda in the A2780 cell line is slightly different, as compared to the cellular processing of cis-[Pt(bapda)Cl2].
As shown in Fig. 6.5, the accumulation of bapda is localized in the cell membrane when imaging after a few minutes, 1 h, or 3 h after the incubation, while for its platinum(II) compound the accumulation is very specific, within acidic lysosomes (Fig. 6.2). This behavior suggests a specific response of the A2780 cell line to the platinum(II) compound. The accumulation of free ligand bapda in the A2780 cell line 24 h after incubation is similar to the one found for cis-[Pt(bapda)Cl2] (images not shown). Thus, the cell responds to the toxic compounds similarly after 24 h, which might be related to the detoxification mechanisms. The same comparison between the free ligand and cis-[Pt(bapda)Cl2] was performed in the A2780R cell line; in this case no differences have been found (images not shown). It must be noted that the present results as such do not prove that platinum(II) is still attached to the bapda ligand in the acidic vesicles. In addition, it is important to mention that the cellular processing studies of both cis-[Pt(bapda)Cl2] and the ligand bapda showed no fluorescence in the cell nucleus in either the A2780 or the A2780R cell lines. This observation may indicate that the anthracene ring intercalates between the DNA base pairs, or that the concentration of the platinum compound in the nucleus is low.
It has been reported that glutathione can easily remove the platinum ion from the alkylenediamine ligands in dinuclear complexes.14 However, in the case of cis-[Pt(bapda)Cl2], the carrier ligand is coordinated in a chelating fashion and has bulky side arms; thus, the stability of this new platinum(II) compound would be higher against GSH inactivation. Moreover, as discussed above, no evidence of in vitro reactivity
between cis-[Pt(bapda)Cl2] and GSH is detected by 195Pt NMR spectroscopy. To investigate whether glutathione facilitates the sequestration of these compounds into the lysosomes as a detoxification cell-mechanism, cellular processing of cis-[Pt(bapda)Cl2] and bapda in GSH-depleted A2780R cells were followed over time. In agreement with the cytotoxic data, no changes in the cellular processing have been found related to cis-[Pt(bapda)Cl2] treatment, where similar lysosomal accumulation could be observed (images not shown) in BSO treated or untreated A2780R cells. However, differences in the cellular processing of free bapda between BSO-treated or untreated A2780R cells have been found, also in agreement with the cytotoxic activity data (Table 6.1), where significant differences between the pEC50 values against the A2780R and the GSH depleted A2780R cells have been observed. The sequestration of the ligand bapda is visible over the cell in some vesicles different than acidic lysosomes (Fig. 6.6 and 6.7), as well as in the lysosomes. This observation suggests that different detoxification mechanisms are involved for cis-[Pt(bapda)Cl2] and for the ligand bapda, with an important role of the GSH levels in the A2780R cells.
A
B
C
Fig. 6.5. Cellular distribution of the ligand bapda compound in the A2780 human ovarian carcinoma cell line. All the images were taken after the incubation period (15 min) has finished. Images A: 10 min after incubation. Images B: 1 h after incubation. Images C: 3 h after incubation. Phase-contrast images are shown on the left, corresponding fluorescent images are in the middle, and superimposed images are on the right.
A
B
Fig. 6.6. Cellular distribution of the ligand bapda 24 h after incubation in A: depleted GSH A2780R cells.
B: in the A2780R cells. Phase-contrast images are shown on the left side, corresponding fluorescence images are in the middle, and superimposed images are on the right side.
A B
C D
Fig. 6.7. Cellular distribution of the ligand bapda compound in GSH- depleted A2780R cells 24 h after incubation. Localization studies with LysoTrackerTM Red DND-99: (A) phase-contrast image of living A2780 superimposed to bapda; (B) bapda fluorescence (blue); (C) staining of lysosomes (red); (D) superimposed representation of the images of bapda (blue) and LysoTrackerTM Red DND-99 (red).
6.3.4. Interaction with 9-ethylguanine
It is generally accepted that the cellular target of the platinum compounds is the DNA, and for this reason the platinum-DNA adduct formation has been related in many occasions to the biological activity of the platinum-based drugs. In particular, cis-[Pt(bapda)Cl2] was allowed to react with 9-ethylguanine (9-EtG) at 37 °C to investigate how this compound would interact with nucleobases. For this interaction a molar ratio of 1:2 was chosen for platinum(II) compound to nucleobases. The platinum compound and 9-EtG were both dissolved in DMF-d7 and 1H NMR spectra were recorded at 37 °C. The reaction between cis-[Pt(bapda)Cl2] and 9-EtG was followed over time. During the first 8 h of reaction no changes could be observed; however, after 10 h small peaks of a new product started to become visible. The reaction with 9-EtG to cis-[Pt(bapda)Cl2] apparently results in the slow formation of a small amount of [Pt(bapda)(9-EtG)Cl]Cl; however, the original cis-[Pt(bapda)Cl2] remains visible in the
1H NMR spectrum after 24 h and 48 h incubation (Fig. 6.8), still being the major species in solution.
Fig. 6.8. Aromatic region of the 1H NMR spectrum (300 MHz) of cis-[Pt(bapda)Cl2] in DMF-d7 at 37 °C reacted with 2 eq. 9-EtG; a: cis-[Pt(bapda)Cl2] in DMF-d7, b: 10 h after mixing, c: 24 h after mixing. New peaks are indicated (arrows). Modification of the H6 and H6’ is indicated with the * symbol.
In addition, the 195Pt NMR spectrum after 24 h of reaction was recorded, now yielding two peaks: one at -2181 ppm corresponding to the unreacted cis-[Pt(bapda)Cl2], and a new small peak at -2335 ppm, which corresponds to the formed platinum-9-EtG
monoadduct product of the reaction (Fig. 6.9), according to the [N3Cl] environment around the platinum(II) ion.20
Fig. 6.9. 195Pt NMR spectrum (300 MHz) of cis-[Pt(bapda)Cl2)] in DMF-d7 at 37 °C reacted with 2 eq.
9-EtG 24 h after mixing.
In addition to NMR spectroscopy, an ESI-MS spectrum of the DMF solution after 24 h of reaction was recorded, showing the formation of the [Pt(bapda)(9-EtG)Cl]+ species, as confirmed by a m/z with a typical platinum isotopic pattern at 864 (Fig. 6.10).
Moreover, a peak at m/z = 686 corresponding to [Pt(bapda)Cl]+ was also observed confirming the presence of both species in solution.
All these observations suggest that the steric bulk of the ligands around the platinum(II) ion indeed hampers the approach of the 9-EtG molecule to the metal center and seriously slows down the reaction.
Nevertheless, cis-[Pt(bapda)Cl2] shows high cytotoxic activity against the human ovarian carcinoma cells (Table 6.1). The poor reactivity to the nucleobase 9-ethylguanine, and the high cytotoxicity of bapda ligand against the same cell lines, suggest that the cytotoxic activity of cis-[Pt(bapda)Cl2] stems largely from the anthracene rings. It has been reported that platinum(II) compounds containing N,N’-diethyl-2,4- pentanediamine (abbreviated as eap) as carrier ligand, such as [Pt(R,R-eap)Cl2] and [Pt(S,S-eap)Cl2] display low cytotoxic activity.21
These compounds were designed to bind enantioselectively to GpG and ApG sequences of DNA, in an attempt to generate enantioselective interactions with DNA, and therefore, enantiospecific cytotoxicity.21 Nevertheless, the cytotoxic activity of these two platinum(II) compounds show only small differences, in contrast to the expectations, against leukemia and human bladder tumor cells.21 The attachment of an anthracene ring
to the two nitrogen atoms of the diamine moiety has increased the biological activities of this class of platinum(II) compounds.
Fig. 6.10. ESI-MS spectrum after 24 h reaction of cis-[Pt(bapda)Cl2] and 2 equivalents of 9-EtG in DMF-d7. Peak at m/z = 864 corresponding to [Pt(bapda)(9-EtG)Cl]+ species.
6.3.5. DNA platination of cis-[Pt(bapda)Cl2]
DNA platination studies of cis-[Pt(bapda)Cl2] and cisplatin as reference compound have been performed to investigate whether the platinum compound interacts with cellular DNA in human ovarian carcinoma cells. The DNA was isolated as described above, and the DNA concentration was determined by measuring the UV absorption at 260 nm. The platinum content was measured by flameless atomic absorption spectroscopy. The results are expressed in nmol of platinum found per million nucleotide and depicted in Fig. 6.11.
As expected for cisplatin treatment, the platinum-DNA adducts levels in the A2780R are significantly lower than the ones formed in the sensitive cell line by cisplatin, as reported previously.22, 23
Surprisingly, cis-[Pt(bapda)Cl2] forms significantly higher levels of platinum- DNA adducts in the resistant cell line, as compared to the sensitive A2780 cells. This observation suggests that DNA-repair mechanisms24 in the A2780R cells might be
activated against the interaction between cis-[Pt(bapda)Cl2] and DNA, since long-time incubation with cis-[Pt(bapda)Cl2] results in cross-resistance to cisplatin. It is also possible that the A2780R cells have high tolerance to the DNA damage caused by cis-[Pt(bapda)Cl2].
0 10 20 30 40 300 600 900
0 10 20 30 40 300 600 900
cisplatin
nmol Pt/million nucleotides
A2780 A2780R
cis-[Pt(bapda)Cl2]
Fig. 6.11. Intracellular DNA platination of cisplatin and cis-[Pt(bapda)Cl2] in the A2780 and the A2780R cells after 1 h incubation at 50 M final concentration (mean ±SD, n = 3)
6.3.6. DNA fluorescence titration
Fluorescence quenching of platinum compounds containing intercalating agents in the presence of DNA has been described in the literature.6 DNA titration experiments displays a clear quenching on the fluorescence emission of cis-[Pt(bapda)Cl2] (ex = 370 nm) with the addition of small amounts of a concentrated calf thymus DNA solution (Fig. 6.12) This observation suggests that cis-[Pt(bapda)Cl2] interacts with DNA primarily via intercalation.
The interaction between ligand bapda and DNA was also studied, giving also a clear quenching of the fluorescence emission upon addition of calf thymus DNA (spectra not shown). As mentioned above, the cytotoxic activity of ligand bapda as compared to cis-[Pt(bapda)Cl2] summarized in Table 6.1 shows that the coordination of the carrier ligand to platinum slightly improves the biological activity. The ligand bapda was found to intercalate to the DNA, as well as its corresponding platinum(II) compound.
Nevertheless, cis-[Pt(bapda)Cl2] presents also the possibility of interacting with the N7 position of the guanine bases (Fig. 6.8), although this interaction is likely to be strongly hindered.
400 450 500 550 600
relative intensity
wavelength (nm)
1:0 2:1 4:1
Fig. 6.12. Quenching of cis-[Pt(bapda)Cl2] fluorescence emission upon titration with calf thymus DNA at various ratios (DNA base pairs : cis-[Pt(bapda)Cl2])
6.4. Concluding remarks and outlook
The cytotoxic assays of both the free ligand and cis-[Pt(bapda)Cl2] show a high antiproliferative activity against a pair of human ovarian carcinoma cell lines, the sensitive line (A2780) and its cisplatin-resistant counterpart (A2780R). The binding of platinum(II) ion in cis-[Pt(bapda)Cl2] improves the activity of bapda, a potential intercalator; however, cross-resistance with cisplatin has been found for cis-[Pt(bapda)Cl2] against the A2780R cells. Inactivation of the platinum(II) compound by GSH does not seem to play an important role as shown by the cytotoxic activity studies with GSH depleted cells, since no significant changes have been observed compared to the A2780R cells. In addition, the reaction between GSH and cis-[Pt(bapda)Cl2] over time has been investigated by 195Pt NMR spectroscopy, which strongly suggests that GSH cannot remove the platinum(II) ion coordinated to the bapda carrier ligand not even after 24 h of reaction.
The interaction of cis-[Pt(bapda)Cl2] with nucleobases was also investigated, which has resulted in the conclusion that steric protection around the platinum(II) ion most likely hampers the 9-EtG approach to the metal ion, giving only a very low rate of interaction. In the literature interesting classes of organic molecules, so-called bisintercalators,25 have been synthesized by linking two heterocycles with chains of
varying lengths between the intercalator moieties.26 These special molecules have been investigated successfully as carrier ligands in the synthesis of new platinum(II) antitumor agents.27 Studies where a quenching of the fluorescence emission of cis-[Pt(bapda)Cl2] due to the interaction with calf thymus DNA has been performed. This quenching strongly suggests that cis-[Pt(bapda)Cl2] interact with DNA also via intercalation.
Moreover, the cellular processing of cis-[Pt(bapda)Cl2] using fluorescence microscopy shows no fluorescent signal in the cell nucleus, suggesting again intercalation of the anthracene ring between the base pairs of the DNA.
Finally, interaction of cis-[Pt(bapda)Cl2] with nuclear DNA in the A2780 and the A2780R cells has been investigated. Higher levels of platinum-DNA adducts have been obtained after 1 h of incubation with cis-[Pt(bapda)Cl2], as compared to those formed by cisplatin. Interestingly, the DNA platination of this new platinum(II) compound in the resistant cells is significantly higher than in the sensitive cells. This suggests that the cross-resistance to cisplatin found after 48 h incubation with cis-[Pt(bapda)Cl2] against the A2780R cells might be due to DNA-repair mechanisms, or due to increased tolerance to DNA-platination in the A2780R cells.
Cellular processing of cis-[Pt(bapda)Cl2] and the ligand bapda shows accumulation of these compounds in lysosomes after short incubation with cis-[Pt(bapda)Cl2], which can be explained by the cross-resistance with cisplatin found for cis-[Pt(bapda)Cl2] cytotoxic data. To investigate whether glutathione facilitates sequestration of cis-[Pt(bapda)Cl2] in lysosomes, fluorescence microscopy in GSH depleted A2780R cells treated with L-BSO has been investigated. Because similar cellular processing in A2780R and GSH-depleted A2780R cells has been observed, it appears that the sequestration of cis-[Pt(bapda)Cl2] after short-time incubation in lysosomes is not related to the GSH levels, indicating a specific drug-uptake pathway.
Instead, a quite different cellular processing has been observed when the cellular processing of the ligand bapda was investigated in GSH-depleted A2780R cells, where an increased number of different vesicles are visible in the cells 24 h after incubation (Fig. 6.7). This suggests that the cellular processing of bapda is influenced by the concentration of GSH, which is also in agreement with its cytotoxic activity against L-BSO treated A2780R and normal A2780R cells. On the other hand, the cellular
distribution of cis-[Pt(bapda)Cl2] and the ligand bapda in the A2780 cells appears to be slightly different. Both compounds accumulate in acidic lysosomes over time; however, the platinum(II) compound is sequestered faster into these vesicles. This observation suggests that the detoxification mechanisms of the A2780 cells respond differently to the platinum(II) compound.
It is believed that platinum-based anticancer drugs target the nuclear DNA, and the sequestration of the platinum drugs in cytoplasmic organelles (trans-Golgi network, endosomes and/or lysosomes) may lead to decreased drug-target interaction and subsequently, to a decreased cytotoxic activity. Cis-[Pt(bapda)Cl2] forms high levels of DNA adducts in the A2780 and the A2780R cells (Fig. 6.11). Therefore, the sequestration of cis-[Pt(bapda)Cl2] in acidic lysosomes does not prevent the formation of large amounts of DNA adducts, which is also shown by the high cytotoxic activity of this compound (Table 6.1).
References
1. Boulikas, T.; Vougiouka, M., Oncol. Rep. 2003, 10, 1663-1682.
2. Reedijk, J., Platinum Metals Rev 2008, 52, 2-11.
3. Molenaar, C.; Teuben, J. M.; Heetebrij, R. J.; Tanke, H. J.; Reedijk, J., J. Biol. Inorg. Chem. 2000, 5, 655-665.
4. Kalayda, G. V.; Wagner, C. H.; Buss, I.; Reedijk, J.; Jaehde, U., BMC Cancer 2008, 8.
5. Safaei, R.; Howell, S. B., Crit. Rev. Oncol./Hematol. 2005, 53, 13-23.
6. Jansen, B. A. J.; Wielaard, P.; Kalayda, G. V.; Ferrari, M.; Molenaar, C.; Tanke, H. J.; Brouwer, J.; Reedijk, J., J. Biol. Inorg. Chem. 2004, 9, 403-413.
7. Kalayda, G. V.; Jansen, B. A. J.; Molenaar, C.; Wielaard, P.; Tanke, H. J.; Reedijk, J., J. Biol.
Inorg. Chem. 2004, 9, 414-422.
8. Kalayda, G. V.; Jansen, B. A. J.; Wielaard, P.; Tanke, H. J.; Reedijk, J., J. Biol. Inorg. Chem.
2005, 10, 305-315.
9. Kalayda, G. V.; Zhang, G. F.; Abraham, T.; Tanke, H. J.; Reedijk, J., J. Med. Chem. 2005, 48, 5191-5202.
10. 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.
11. Alderden, R. A.; Mellor, H. R.; Modok, S.; Hambley, T. W.; Callaghan, R., Biochem. Pharmacol.
2006, 71, 1136-1145.
12. Heffeter, P.; Jungwirth, U.; Jakupec, M.; Hartinger, C.; Galanski, M.; Elbling, L.; Micksche, M.;
Keppler, B.; Berger, W., Drug Resist. Update 2008, 11, 1-16.
13. Martelli, L.; Di Mario, F.; Ragazzi, E.; Apostoli, P.; Leone, R.; Perego, P.; Fumagalli, G., Biochem. Pharmacol. 2006, 72, 693-700.
14. Jansen, B. A. J.; Brouwer, J.; Reedijk, J., J. Inorg. Biochem. 2002, 89, 197-202.
15. Wheate, N. J.; Brodie, C. R.; Collins, J. G.; Kemp, S.; Aldrich-Wright, J. R., Mini-Rev. Med.
Chem. 2007, 7, 627-648.
16. Mosmann, T., J. Immunol. Methods 1983, 65, 55-63.
17. Mistry, P.; Kelland, L. R.; Abel, G.; Sidhar, S.; Harrap, K. R., Br. J. Cancer 1991, 64, 215-220.
18. Goto, S.; Yoshida, K.; Morikawa, T.; Urata, Y.; Suzuki, K.; Kondo, T., Cancer Res. 1995, 55, 4297-4301.
19. Merlin, J. L.; Bour-Dill, C.; Marchal, S.; Ramacci, C.; Poullain, M. G.; Giroux, B., Cytometry 2000, 41, 62-72.
20. Pregosin, P. S., Coord. Chem. Rev. 1982, 44, 247-291.
21. Vickery, K.; Bonin, A. M.; Fenton, R. R.; Omara, S.; Russell, P. J.; Webster, L. K.; Hambley, T.
W., J. Med. Chem. 1993, 36, 3663-3668.
22. Johnson, S. W.; Perez, R. P.; Godwin, A. K.; Yeung, A. T.; Handel, L. M.; Ozols, R. F.; Hamilton, T. C., Biochem. Pharmacol. 1994, 47, 689-697.
23. Zisowsky, J.; Koegel, S.; Leyers, S.; Devarakonda, K.; Kassack, M. U.; Osmak, M.; Jaehde, U., Biochem. Pharmacol. 2007, 73, 298-307.
24. Jung, Y. W.; Lippard, S. J., Chem. Rev. 2007, 107, 1387-1407.
25. Dawson, S.; Malkinson, J. P.; Paumier, D.; Searcey, M., Nat. Prod. Rep. 2007, 24, 109-126.
26. Becker, M. M.; Dervan, P. B., J. Am. Chem. Soc. 1979, 101, 3664-3666.
27. Perez, J. M.; Lopez-Solera, I.; Montero, E. I.; Brana, M. F.; Alonso, C.; Robinson, S. P.; Navarro- Ranninger, C., J. Med. Chem. 1999, 42, 5482-5486.
