# The In Vitro Antitumour Activity of Novel, Mitochondrial-Interactive, Gold-Based Lipophilic Cations

Authors: Sherika Mahepal; Richard Bowen; Messai Adenew Mamo; Marcus Layh; Constance Elizabeth Jansen van Rensburg
Journal: Metal-Based Drugs
 (2008)

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## Abstract

In this study we compared the effects of two previously described
antimitochondrial gold complexes, that is, [A] [Au(dppe)2]Cl
and [B] [Au(d4pype)2]Cl with two novel lipophilic cations,
that is, [C] [Au(dpmaaH2)(dpmaaSnMe2)]Cl and [D]
[Au(dpmaaSnMe2)2]Cl as antimitochondrial agents. The
results of this study indicate that [C] and [D] have intermediate
partition coefficients and exhibited a selective uptake by cells.
They exhibited a higher selectivity for the various cell lines than
[A] but were more cytotoxic than [B]. There is a significant
correlation between the cytotoxic potential of [A], [B], [C], and
[D] and their octanol/water partition coefficients in both MCF-7
(breast cancer) and MCF-12A (nonmalignant breast) cells, whereas
their cytotoxic potential and ability to induce the release of
cytochrome c correlated only in the case of the MCF-12A cells.
Complexes [C] and [D] are promising new chemotherapeutic drugs.
These compounds target the mitochondrial membranes of certain
cancer cells exploiting the differences between the mitochondrial
membrane potential of these cells and normal cells.  Although the
concentrations of these compounds necessary to eradicate cancer
cells are very high, the results provide a basis for the synthesis
of a new family of compounds with intermediate partition
coefficients compared to [A] and [B] but with increased activity
against cancer cells.


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## Body

## 1. Introduction

The successful chemotherapy of  cancer has been
hindered by the acquired resistance of tumour cells to clinical drugs, which
has necessitated the use of multidrug therapy with a higher potential for
adverse drug effects.  The use of
relatively nonselective compounds results in the eradication of normal cells in
addition to tumour cells.  Previous
studies have indicated that[Au(dppe)2]Cl (see Figure 1) exhibits
antitumour activity in a wide range of tumour models in mice [1].  Since the lipophilic cationic properties of
[Au(dppe)2]Cl promote its nonselective uptake into the mitochondria
of all cells, strategies were adopted to synthesize more hydrophilic analogues
that retained antitumour activity while being less toxic to the
mitochondria.Structure of the experimental compounds:[Au(dppe)2]Cl [A],  [Au(d4pype)2]Cl [B],  [Au(dpmaaSnMe2)(dpmaaH2)]Cl [C], and [Au(dpmaaSnMe2)2]Cl [D]. (50 μM).
[A][B][C][D]The lipophilicity of gold(I) phosphine complexes can
be controlled by an appropriate substitution at the quaternary centre or alkyl
backbone that enables the modification of the compound to achieve greater
selectivity for tumour cells [2]. This
has led to the synthesis of the 3- and 4-pyridyl substituted analogues of
[Au(dppe)2]Cl by the replacement of some or all of the phenyl
substituents in dppe with hydrophilic pyridyl groups [3].  The lipophilicity of these tetrahedral
bis(dipyridylphosphino) gold(I) complexes plays a key role in determining their
cellular uptake [4].  It is therefore
necessary to retain the lipophilicity of the compound within a certain
octanol-water coefficient range in order to facilitate the uptake of a drug and
determine the degree of protein binding [2].Although the precise mechanism of action of these
gold-containing lipophilic aromatic compounds is poorly understood, several
studies have suggested a mitochondrial mode of action as well as the occurrence
of DNA strand breaks and DNA-protein crosslinks in tumour cells [1, 2, 4, 5].Tumour cells possess one of the highest mitochondrial
transmembrane potentials [6, 7], which may be due to the inability of tumour
cells to use mitochondria to fulfil their demands for ATP.  Normal cells rely on oxygen consumption and
oxidative phosphorylation for ATP production that takes place in the
mitochondria, whereas tumour cells rely primarily on glycolysis for ATP
production that takes place in the cytosol of the cell [4].  Lipophilic cations that possess a positive
charge are able to penetrate the hydrophobic barriers of the plasma and
mitochondrial membranes [8]. These compounds accumulate in the mitochondria in
response to a negative transmembrane potential, which is the rationale behind
the use of lipophilic cations as anticancer agents.According to Davis et al.  the uptake of a
lipophilic cation is dependent on both the plasma and mitochondrial membrane
potentials. The plasma membrane preconcentrates the drug in the cytoplasm and
the mitochondrial membrane, in turn, concentrates the drug within the
mitochondria [9].  These facts provide
the perfect opportunity for drug accumulation in tumour cells and the
feasibility for the use of tumour selective antimitochondrial compounds in
human cancer chemotherapy.The[Au(d4pype)2]Cl complex (see Figure 1),
which is hydrophilic, exhibited a higher tumour uptake and a smaller in vivo
liver/tumour ratio in treated mice with advanced Colon 38 adenocarcinoma
tumours than [Au(dppe)2]Cl [10]. McKeage et al. [4] corroborated that compounds with an intermediate
lipophilicity when compared to more lipophilic or hydrophilic compounds  displayed significant antitumour activity,
less dose-limiting toxicity, and a higher plasma concentration of gold.  A correlation between the lipophilicity of
the drug and the degree of selectivity and cytotoxic potency of the compounds
has been established [10].  This denotes
that an enhanced selectivity is achieved with greater hydrophilicity but an
increase in the potency of the drug can be observed with a greater lipophilicity.The common
occurrence of drug-resistant tumour cells and the lack of selectivity of cancer
drugs in differentiating between tumour cells and normal cells are two
overriding problems in cancer chemotherapy [10].  In an attempt to combat this lack of
selectivity, an investigation was initiated into two new aromatic cations, that
is, [Au(dpmaaSnMe2)(dpmaaH2)]Cl [C] and [Au(dpmaaSnMe2)2]Cl [D] (see Figure 1).  The simultaneous
presence of gold and tin in aromatic cations should offer the advantage of
reducing the development of resistance in cancer cells.


## 2. Materials and Method

### 2.1. Octanol/Water Partition Coefficient

60μM stock solutions of [Au(dppe)2]Cl [A], Au(d4pype)2]Cl [B], [Au(dpmaaSnMe2)(dpmaaH2)]Cl [C], and [Au(dpmaaSnMe2)2]Cl [D] were
prepared in octanol (Merck, Darmstadt, Germany).   Water-saturated octanol and octanol-saturated water were prepared by shaking
equal volumes of octanol and water for 15 minutes and allowing the mixture to
separate into the respective phases for 20 hours.  The absorbance of the initial drug
concentration in the water-saturated octanol at 20 μM, 40 μM, and 60 μM was
analysed by UV spectrophotometry.  Five
millilitres of the octanol-saturated water were then added to 5 ml of the
drug/octanol solutions to obtain a final volume of 10 ml.  These solutions were shaken vigorously for 15
minutes,   thereafter they were left to separate into an octanol and aqueous phase for
20 hours.  The aqueous phase was
separated ensuring that there was no contamination from the octanol phase, and
each of these solutions was analysed by UV spectrophotometry in 1 cm quartz
cuvettes to obtain the absorbance of the compounds.


### 2.2. Cell Cultures

Cytotoxicity assays were performed on the following
cell cultures: human ovarian
carcinoma (A2780)(ECACC93112519) and its cisplatin-resistant subline
(A2780cis)(ECACC93112517), breast carcinoma (MCF-7)(HTB-22), colon cancer (CoLo
320 DM)(CCL-220), cervical carcinoma cells (HeLa)(CCL-2), mouse melanoma
(B16)(ECACC92101203), primary human fibroblasts (CCL-171), and a breast nontumourogenic
cell line (MCF-12A)(CRL-10782). A2780, A2780cis and CoLo 320 DM were maintained
in RPMI; B16, HeLa cells, and the primary fibroblasts were maintained in
EMEM.  B16 cells were maintained in DMEM.  MCF-7 cells were maintained in DMEM supplemented
with 2% nonessential amino acids and MCF-12A cells were maintained in a1:1
mixture of DMEM and HAMS F12 medium with hydrocortisone (10 mg/mL), cholera
toxin (1 mg/mL), insulin (20 mg/mL), 10% foetal calf serum (FCS), and epidermal
growth factor (100 mg/mL). All media were supplemented with 1% solution of penicillin and streptomycinand
10% heat inactivated FCS.


### 2.3. Cell Growth Assay

A metabolic assay based on the reactivity of MTT
(3-[4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide), originally
described by Mosmann [11] with modifications [12], was used to determine the effects of the
experimental compounds on cell growth after a 7-day treatment period in 96 well-round
bottom microtiter plates.


### 2.4. Cellular Uptake of Gold

MCF-7 and MCF-12A cells were prepared in supplemented
DMEM at a stock concentration of5×106 cells/mL.  The cells were exposed to the various
compounds at concentrations that lead to 80–90% of cell death in the above-mentioned
assay for 30 minutes at 37°C, centrifuged for 10 minutes at 185 g, and the pellets washed with Hank's balanced salt
solution (HBSS). The gold content of the pellets was determined on an
inductively coupled plasma mass spectrometer (ICP-MS) using standard
procedures.


### 2.5. Release of Cytochrome c

A suspension of MCF-7 and MCF-12A cells (2×106 cells/mL)
was prepared in supplemented DMEM.  The
cells were exposed to a concentration of the various compounds that lead to
80–90% of cell death for 30 minutes at 37°C, centrifuged for 10 minutes at 185 g, and the pellets washed with HBSS and assayed according
to the manufacturers’ instructions for
cytochrome c using an assay kit (Sigma Chemical Co, St. Louis, Mo, USA).


### 2.6. Statistical Analysis

The Pearson correlation coefficient (±95% confidence
interval) was calculated and used to determine correlations between the various
assay results.


## 2.1. Octanol/Water Partition Coefficient

60μM stock solutions of [Au(dppe)2]Cl [A], Au(d4pype)2]Cl [B], [Au(dpmaaSnMe2)(dpmaaH2)]Cl [C], and [Au(dpmaaSnMe2)2]Cl [D] were
prepared in octanol (Merck, Darmstadt, Germany).   Water-saturated octanol and octanol-saturated water were prepared by shaking
equal volumes of octanol and water for 15 minutes and allowing the mixture to
separate into the respective phases for 20 hours.  The absorbance of the initial drug
concentration in the water-saturated octanol at 20 μM, 40 μM, and 60 μM was
analysed by UV spectrophotometry.  Five
millilitres of the octanol-saturated water were then added to 5 ml of the
drug/octanol solutions to obtain a final volume of 10 ml.  These solutions were shaken vigorously for 15
minutes,   thereafter they were left to separate into an octanol and aqueous phase for
20 hours.  The aqueous phase was
separated ensuring that there was no contamination from the octanol phase, and
each of these solutions was analysed by UV spectrophotometry in 1 cm quartz
cuvettes to obtain the absorbance of the compounds.


## 2.2. Cell Cultures

Cytotoxicity assays were performed on the following
cell cultures: human ovarian
carcinoma (A2780)(ECACC93112519) and its cisplatin-resistant subline
(A2780cis)(ECACC93112517), breast carcinoma (MCF-7)(HTB-22), colon cancer (CoLo
320 DM)(CCL-220), cervical carcinoma cells (HeLa)(CCL-2), mouse melanoma
(B16)(ECACC92101203), primary human fibroblasts (CCL-171), and a breast nontumourogenic
cell line (MCF-12A)(CRL-10782). A2780, A2780cis and CoLo 320 DM were maintained
in RPMI; B16, HeLa cells, and the primary fibroblasts were maintained in
EMEM.  B16 cells were maintained in DMEM.  MCF-7 cells were maintained in DMEM supplemented
with 2% nonessential amino acids and MCF-12A cells were maintained in a1:1
mixture of DMEM and HAMS F12 medium with hydrocortisone (10 mg/mL), cholera
toxin (1 mg/mL), insulin (20 mg/mL), 10% foetal calf serum (FCS), and epidermal
growth factor (100 mg/mL). All media were supplemented with 1% solution of penicillin and streptomycinand
10% heat inactivated FCS.


## 2.3. Cell Growth Assay

A metabolic assay based on the reactivity of MTT
(3-[4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide), originally
described by Mosmann [11] with modifications [12], was used to determine the effects of the
experimental compounds on cell growth after a 7-day treatment period in 96 well-round
bottom microtiter plates.


## 2.4. Cellular Uptake of Gold

MCF-7 and MCF-12A cells were prepared in supplemented
DMEM at a stock concentration of5×106 cells/mL.  The cells were exposed to the various
compounds at concentrations that lead to 80–90% of cell death in the above-mentioned
assay for 30 minutes at 37°C, centrifuged for 10 minutes at 185 g, and the pellets washed with Hank's balanced salt
solution (HBSS). The gold content of the pellets was determined on an
inductively coupled plasma mass spectrometer (ICP-MS) using standard
procedures.


## 2.5. Release of Cytochrome c

A suspension of MCF-7 and MCF-12A cells (2×106 cells/mL)
was prepared in supplemented DMEM.  The
cells were exposed to a concentration of the various compounds that lead to
80–90% of cell death for 30 minutes at 37°C, centrifuged for 10 minutes at 185 g, and the pellets washed with HBSS and assayed according
to the manufacturers’ instructions for
cytochrome c using an assay kit (Sigma Chemical Co, St. Louis, Mo, USA).


## 2.6. Statistical Analysis

The Pearson correlation coefficient (±95% confidence
interval) was calculated and used to determine correlations between the various
assay results.


## 3. Results

### 3.1. Octanol/Water Partition Coefficient

The average octanol/water partition coefficient values
and the mean log octanol/water partition coefficient values are summarised in Table1.  The results indicate that [A] has a mean log
octanol/water partition coefficient of 1.07, making it a lipophilic compound
whereas [B] is hydrophilic with a very low log octanol/water partition
coefficient of −1.57.  The octanol/water
partition coefficients of both [C] and [D] (−0.012 and 0.09, resp.) are
intermediate to that of [A] and [B].Table 1
Average octanol/water partition
coefficients of the experimental compounds as well as the mean log
octanol/water partition coefficient± standard
error mean (SEM).
Octanol/water partition coefficient andmean log octanol/water partition coefficient± SEMExperimentalAverage1 octanol/waterMean log octanol/watercompoundspartition coefficientpartition coefficientvalue± SEM[A]12.06−1.07±0.1041[B]0.038−1.568±0.149[C]2.3−0.012±0.317[D]1.67−0.09±0.15511 Average of 9 experiments at three
different concentrations.


### 3.2. Cytotoxicity Assays

The IC50 values of the compounds for the
various cell lines are summarised in Table 2.  All eight cell cultures were
more sensitive to [A] than any of the other compounds tested. [B] was more
selective for certain cell types such as MCF-7.
This cell line was 8-9 times more sensitive for this compound than the
colon cancer cell line (CoLo 320DM) and the primary fibroblast culture.  [C] and [D]
also exhibited some selectivity for certain cell types viz [D] acted on
the B16 cells at a concentration that was 9.5 times lower than what was necessary
to inhibit the growth of CoLo 320DM.Table 2
Mean drug concentration (μM) causing 50% cell death (IC50)
after treatment of various cell lines with the five experimental compounds.
Cell lines[A]1[B][C][D]MCF-72.3120.3275.336.508MCF-12A2.42416025.63916.58A27801.15822.24412.99617.277A2780cis2.5837.6631020HeLa1.40763.79311.47612.264CoLo7.68620031.14126.234Primary fibroblasts0.88717011.8748.046B160.11549.894.9992.7381 Average of 3–5 experiments.


### 3.3. Cellular Uptake of Experimental Compounds

The average amount of gold taken up by MCF-7 and
MCF-12A cells after treatment with the various experimental compounds is
summarised in Table3.  The cells treated
with [A] and [B] accumulated almost twice the percentage of gold than cells treated
with [C] and [D].Table 3
Gold uptake (mg/L) by MCF-7 and MCF-12 A cells treated with the
experimental compounds.
Gold Uptake (mg/L)1Cell  lines[A][B][C][D]MCF-75.275.192.852.68MCF-12A6.516.833.32.631 Average of 3 different experiments.


### 3.4. Release of Cytochrome c

The average percentage of cytochrome c release by
MCF-7 and MCF-12A cells is summarised in Table4.  The results indicate that the outer membrane
of mitochondria of cells treated with [A] incurred the greatest amount of
damage as only 2-3% of the cells had undamaged mitochondrial membranes after
treatment.  There were no significant
differences between the sensitivity of the mitochondrial membranes of MCF-7A
and MCF-12A for the experimental drugs.Table 4
Mitochondria of MCF-7 and MCF-12A cells with undamaged membranes after treatment with experimental compounds.
Percentage mitochondria with undamaged
membranescalculated as the invert of the percentage cytochrome c release1Cell lineUntreated[A][B][C][D]MCF-760.75361.7539.541.0MCF-12A70.252.2576.548.046.251Average of 3–5 experiments.


### 3.5. Pearson’s Correlation Coefficient

(i) There is a significant
correlation (P<.05) between the cytotoxic potential of [A], [B],
[C], and [D] and their octanol/water partition coefficients in both MCF-7 and
MCF-12A cells.(ii) There is a significant
correlation (P<.05) between the cytotoxic potential of [A], [B],
[C] and [D] and their ability to induce the release of cytochrome c in MCF-12A
cells.


## 3.1. Octanol/Water Partition Coefficient

The average octanol/water partition coefficient values
and the mean log octanol/water partition coefficient values are summarised in Table1.  The results indicate that [A] has a mean log
octanol/water partition coefficient of 1.07, making it a lipophilic compound
whereas [B] is hydrophilic with a very low log octanol/water partition
coefficient of −1.57.  The octanol/water
partition coefficients of both [C] and [D] (−0.012 and 0.09, resp.) are
intermediate to that of [A] and [B].Table 1
Average octanol/water partition
coefficients of the experimental compounds as well as the mean log
octanol/water partition coefficient± standard
error mean (SEM).
Octanol/water partition coefficient andmean log octanol/water partition coefficient± SEMExperimentalAverage1 octanol/waterMean log octanol/watercompoundspartition coefficientpartition coefficientvalue± SEM[A]12.06−1.07±0.1041[B]0.038−1.568±0.149[C]2.3−0.012±0.317[D]1.67−0.09±0.15511 Average of 9 experiments at three
different concentrations.


## 3.2. Cytotoxicity Assays

The IC50 values of the compounds for the
various cell lines are summarised in Table 2.  All eight cell cultures were
more sensitive to [A] than any of the other compounds tested. [B] was more
selective for certain cell types such as MCF-7.
This cell line was 8-9 times more sensitive for this compound than the
colon cancer cell line (CoLo 320DM) and the primary fibroblast culture.  [C] and [D]
also exhibited some selectivity for certain cell types viz [D] acted on
the B16 cells at a concentration that was 9.5 times lower than what was necessary
to inhibit the growth of CoLo 320DM.Table 2
Mean drug concentration (μM) causing 50% cell death (IC50)
after treatment of various cell lines with the five experimental compounds.
Cell lines[A]1[B][C][D]MCF-72.3120.3275.336.508MCF-12A2.42416025.63916.58A27801.15822.24412.99617.277A2780cis2.5837.6631020HeLa1.40763.79311.47612.264CoLo7.68620031.14126.234Primary fibroblasts0.88717011.8748.046B160.11549.894.9992.7381 Average of 3–5 experiments.


## 3.3. Cellular Uptake of Experimental Compounds

The average amount of gold taken up by MCF-7 and
MCF-12A cells after treatment with the various experimental compounds is
summarised in Table3.  The cells treated
with [A] and [B] accumulated almost twice the percentage of gold than cells treated
with [C] and [D].Table 3
Gold uptake (mg/L) by MCF-7 and MCF-12 A cells treated with the
experimental compounds.
Gold Uptake (mg/L)1Cell  lines[A][B][C][D]MCF-75.275.192.852.68MCF-12A6.516.833.32.631 Average of 3 different experiments.


## 3.4. Release of Cytochrome c

The average percentage of cytochrome c release by
MCF-7 and MCF-12A cells is summarised in Table4.  The results indicate that the outer membrane
of mitochondria of cells treated with [A] incurred the greatest amount of
damage as only 2-3% of the cells had undamaged mitochondrial membranes after
treatment.  There were no significant
differences between the sensitivity of the mitochondrial membranes of MCF-7A
and MCF-12A for the experimental drugs.Table 4
Mitochondria of MCF-7 and MCF-12A cells with undamaged membranes after treatment with experimental compounds.
Percentage mitochondria with undamaged
membranescalculated as the invert of the percentage cytochrome c release1Cell lineUntreated[A][B][C][D]MCF-760.75361.7539.541.0MCF-12A70.252.2576.548.046.251Average of 3–5 experiments.


## 3.5. Pearson’s Correlation Coefficient

(i) There is a significant
correlation (P<.05) between the cytotoxic potential of [A], [B],
[C], and [D] and their octanol/water partition coefficients in both MCF-7 and
MCF-12A cells.(ii) There is a significant
correlation (P<.05) between the cytotoxic potential of [A], [B],
[C] and [D] and their ability to induce the release of cytochrome c in MCF-12A
cells.


## 4. Discussion

Results from this study have indicated that [B], [C],
and [D] have octanol/water partition coefficients that are lower than that of [A].  These compounds exhibit more selectivity for
different cell types but are less intrinsically potent.  The lower octanol/water partition
coefficients facilitate the more selective uptake of these compounds.  On the other hand, Berners-Price et. al. [13]
described a related compound, that is, [Au(dpmaaH2)2]Cl, to be hydrophilic with no significant
activity against cancer cells indicating the important role lypophilicity plays
in the  design of antitumour compounds.A significant correlation between drug uptake and the
octanol/water partition coefficient of compounds has been established
[10].  Although [C] and [D] have
intermediate partition coefficients we failed to obtain a significant
correlation between drug uptake and the octanol/water partition
coefficient.McKeage et al. [4] affirmed that compounds with an intermediate lipophilicity displayed
significant antitumour activity, less dose-limiting toxicity, and a higher
plasma concentration of gold when compared to more lipophilic or hydrophilic
compounds.  Similarly, we found a
significant correlation between lipophilicity and cytotoxicity.  However, results from this study suggest that
the cellular uptake of these compounds is not dependent on the lipophilicity of
the compound.The mere fact that cytochrome c is being released
indicates that the cell will eventually undergo apoptosis.  The uncoupling of oxidative phosphorylation
results in the swelling of the mitochondria [5], which consequently causes the
outer membrane of the mitochondria to rupture, leading to the release of
cytochrome c [7].  In this study, we
found a significant correlation between lipophilicity and mitochondrial damage
only in the case of the nontumourogenic breast cell line (MCF-12A) and not the
breast cancer cell line (MCF-7) indicating a possible selectivity of less
lipophilic compounds for mitochondrial cell membranes.Furthermore, cytotoxicity results indicate that MCF-7
is more sensitive than MCF-12A to [B], [C], and [D].The results from this study suggest that [C] and [D]
act more selectively against a breast cancer and myeloma cell line than [B] but
possess less overall cytotoxicity compared to [A], which is an important
characteristic in selecting anticancer agents.
These compounds possess an intermediate partition coefficient compared
to [A] and [B], which plays an important role in their uptake by both normal
and cancer cells.  Future work will
include experimental studies to obtain a clear understanding of the mechanism
of action of [C] and [D] and the influence of tin in this regard.  Although the high IC50 values
obtained with [C] and [D] against malignant cell lines are not achievable in vivo, it provides a basis for the
synthesis of a new family of compounds with intermediate partition coefficients
compared to [A] and [B] but increased activity against cancer cells.



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*Source: 864653-2008-03-27.xml*