Pt-Mal-LHRH, a Newly Synthesized Compound Attenuating Breast Cancer Tumor Growth and Metastasis by Targeting Overexpression of the LHRH Receptor

Article abstract of DOI:10.1021/acs.bioconjchem.6b00610

A new targeting chemotherapeutic agent, Pt-Mal-LHRH, was synthesized by linking activated cisplatin to luteinizing hormone releasing hormone (LHRH). The compound's efficacy and selectivity toward 4T1 breast cancer cells were evaluated. Carboplatin was selected as the comparative platinum complex, since the Pt-Mal-LHRH malonate linker chelates platinum in a similar manner to carboplatin. Breast cancer and normal cell viability were analyzed by an MTT assay comparing Pt-Mal-LHRH with carboplatin. Cells were also treated with either Pt-Mal-LHRH or carboplatin to evaluate platinum uptake by ICP-MS and cell migration using an in vitro scratch-migration assay. Tumor volume and metastasis were evaluated using an in vivo 4T1 mouse tumor model. Mice were administered Pt-Mal-LHRH (carboplatin molar equivalent dosage) through ip injection and compared to those treated with carboplatin (5 (mg/kg)/week), no treatment, and LHRH plus carboplatin (unbound) controls. An MTT assay showed a reduction in cell viability (p < 0.01) in 4T1 and MDA-MB-231 breast cancer cells treated with Pt-Mal-LHRH compared to carboplatin. Pt-Mal-LHRH was confirmed to be cytotoxic by flow cytometry using a propidium iodide stain. Pt-Mal-LHRH displayed a 20-fold increase in 4T1 cellular uptake compared to carboplatin. There was a decrease (p < 0.0001) in 4T1 cell viability compared to 3T3 normal fibroblast cells. Treatment with Pt-Mal-LHRH also resulted in a significant decrease in cell-migration compared to carboplatin. In vivo testing found a significant reduction in tumor volume (p < 0.05) and metastatic tumor colonization in the lungs with Pt-Mal-LHRH compared to carboplatin. There was a slight decrease in lung weight and no difference in liver weight between treatment groups. Together, our data indicate that Pt-Mal-LHRH is a more potent and selective chemotherapeutic agent than untargeted carboplatin.

Full text of DOI:10.1021/acs.bioconjchem.6b00610

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Article
Pt-Mal-LHRH, a Newly Synthesized Compound
Attenuating Breast Cancer Tumor Growth and Metastasis
by Targeting Overexpression of the LHRH Receptor
Lindsay E Calderon, Jonathan K Keeling, Joseph D Rollins, Carrie A Black,
Kendall Collins, Nova L Arnold, Diane E. Vance, and Margaret W Ndinguri
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00610 • Publication Date (Web): 07 Dec 2016
Downloaded from http://pubs.acs.org on December 7, 2016
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Pt-Mal-LHRH, a Newly Synthesized Compound Attenuating Breast Cancer
Tumor Growth and Metastasis by Targeting Overexpression of the LHRH
Receptor
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Lindsay E. Calderon†*, Jonathan K. Keeling‡, Joseph Rollins†, Carrie A. Black‡, Kendall Collins†, Nova
Arnold†, Diane E. Vance‡, Margaret W. Ndinguri‡*
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†Department of Biology, Eastern Kentucky University, Richmond, KY, 40475, USA
‡Department of Chemistry, Eastern Kentucky University, Richmond, KY, 40475, USA
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*Corresponding Authors: Margaret W. Ndinguri: Margaret.Ndinguri@eku.edu and Lindsay
E. Calderon: Lindsay.Calderon@eku.edu
New Science Building 4126
521 Lancaster Avenue
Richmond KY 40475
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Abstract
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A new targeting chemotherapeutic agent, PtꢀMalꢀLHRH, was synthesized by linking activated cisplatin to
luteinizing hormone releasing hormone (LHRH). The compound’s efficacy and selectivity towards 4T1
breast cancer cells were evaluated. Carboplatin was selected as the comparative platinum complex since
the PtꢀMalꢀLHRH malonate linker chelates platinum in a similar manner to carboplatin. Breast cancer
and normal cell viability were analyzed by an MTT assay comparing PtꢀMalꢀLHRH with
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carboplatin. Cells were also treated with either PtꢀMalꢀLHRH or carboplatin to evaluate platinum uptake
by ICPꢀMS and cell migration using an in-vitro scratchꢀmigration assay. Tumor volume and metastasis
were evaluated using an in-vivo 4T1 mouse tumor model. Mice were administered PtꢀMalꢀLHRH
(carboplatin molarꢀequivalent dosage) through IP injection and compared to those treated with carboplatin
(5 mg/kg/wk), no treatment, and LHRH plus carboplatin (unbound) controls. An MTT assay showed a
reduction in cell viability (p<0.01) in 4T1 and MDAꢀMBꢀ231 breast cancer cells treated with PtꢀMalꢀ
LHRH compared to carboplatin. PtꢀMalꢀLHRH was confirmed to be cytotoxic by flow cytometry using a
propidium iodide stain. PtꢀMalꢀLHRH displayed a 20ꢀfold increase in 4T1 cellular uptake compared to
carboplatin. There was a decrease (p<0.0001) in 4T1 cell viability compared to 3T3 normal fibroblast
cells. Treatment with PtꢀMalꢀLHRH also resulted in a significant decrease in cellꢀmigration compared to
carboplatin. In-vivo testing found a significant reduction in tumor volume (p<0.05) and metastatic tumor
colonization in the lungs with PtꢀMalꢀLHRH compared to carboplatin. There was a slight decrease in
lung weight and no difference in liver weight between treatment groups. Together, our data indicates that
PtꢀMalꢀLHRH is a more potent and selective chemotherapeutic agent than untargeted carboplatin.
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Introduction
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In the United States breast cancer afflicts 1 in 8 women during their lifetime.1 The National
Cancer Institute’s SEER database indicates the median overall survival rate for stage IV breast cancer is
23%. Despite recent advancements in the firstꢀline treatments, many patients eventually relapse, their
tumors become chemoresistant, and patients experience numerous debilitating side effects from
chemotherapy treatment. Major issues of chemotherapy administration stem from the resulting severe side
effects, which causes doctors to often resort to dose reduction, treatment delay or suspension of therapy.
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The platinum drugs carboplatin and cisplatin have been used to treat breast, prostate, ovarian,
bladder, lung, head and neck cancer.2ꢀ3 Cisplatin is the most potent member of the platinum family;
however, due to its poor targeting and formation of resistance, it is not an effective treatment option for
breast cancer.4ꢀ5 Carboplatin, an analogue of cisplatin has gained greater use due to its lower nonꢀspecific
toxicity and higher activity against cisplatinꢀresistant tumors.6 Platinum anticancer drugs promote cell
death by interfering with cellular transcription and DNA replication mechanisms. Serious side effects
resulting from the platinum family drugs include nephrotoxicity, myelotoxicity, neurotoxicity, vomiting
and nausea.7 Another possible concern is the ability of certain cancers to develop resistance mechanisms
to cisplatin including efflux pumps.8 To combat acquired and intrinsic multidrug resistance, as well the
severe side effects exhibited during high dose treatment, recent cancer research is focused on developing
targeted cancer delivery to decrease side effects while improving efficacy.
Systemic side effects result from anticancer drugs which are primarily designed to destroy rapidly
dividing cells, including those found in healthy tissues. To decrease adverse side effects and limit
nonspecific activity several methods of receptor directed chemotherapy have been developed using
receptor ligands, lectins, antibodies, sugars, hormones and hormone analogs.9ꢀ14 Luteinizing hormone
releasing hormone (LHRH) receptor, also referred to as gonadotropin releasing hormone (GnRH)
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receptor, has been found to be overexpressed in breast, prostate, endometrial and ovarian cancers in
comparison to normal cells. This makes the LHRH peptide a good candidate for drug targeting.15ꢀ17
In many of these cancers, tumor proliferation can be inhibited by antagonist analogues of
LHRH.18ꢀ19 Previous studies have demonstrated that cytotoxic compound attachment at the Dꢀlys moiety
of LHRH does not change the intrinsic high binding affinity of the peptide to its receptors.14, 20ꢀ21 For
example, Schally and Nagy studied the potent antagonist on conjugates of [DLys6]ꢀLHRH–DOX and
[DLys6]ꢀLHRH–2ꢀpyrrolinoꢀDOX revealing increased efficacy of doxorubicin.22 The LHRH analogue
maintained its high targeted binding affinity while the drug retained its cytotoxic effects on the tumor
cells.14 Aggarwal and coworkers showed that [DLys6]ꢀLHRH–curcumin enhanced apoptosis of tumor
tissue and was useful in the treatment of pancreatic cancer.12 In another study, Hansel and coworkers
showed that conjugates of lytic peptides and LHRH are very effective in destroying human breast and
prostate cancer xenografts that express LHRH receptors.23ꢀ26 Mudyiwa et al. also synthesized a [DLys6]ꢀ
LHRH conjugate with phthalocyanine; however, the results of the biological testing have not been
released.27
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Although numerous breast cancers overexpress LHRH receptors compared to normal breast cells,
few findings have been published regarding the role of LHRH and platinum drugs. This paper
demonstrates the chemical synthesis of the new compound PtꢀMalꢀLHRH by attaching LHRH (targeting
moiety) with activated cisplatin and reports data on the potency and selectivity of this chemotherapeutic
agent towards breast cancer. Carboplatin was selected as the comparative platinum complex since the Ptꢀ
MalꢀLHRH malonate linker chelates platinum in a similar manner to carboplatin (Figure 1).
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Figure 1: Chemical structure of Pt-Mal-LHRH and carboplatin. The bioactive region/structure of
carboplatin is maintained in the Pt-Mal-LHRH compound.
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Synthesis of Pt-Mal-LHRH
For complete synthesis of PtꢀMalꢀLHRH, a malonate linker was first synthesized which contains
a unique tumorꢀactivated protease cleavage site.12 Diꢀtertꢀbutyl 2ꢀ(3ꢀ(4ꢀCarboxybutanamido)propyl)ꢀ
malonate (Mal) was synthesized as described in Ndinguri et al.13 (Figure 1). Use of the malonate linker is
advantageous as it will allow the platinum drug to be bioactive in the tumor cells. Starting from an amide
based resin and using Fmoc chemistry, the peptide was assembled, the Alloc protecting group was
removed followed by conjugation of the Mal. The fully assembled peptide was cleaved from the solid
support using a cleavage cocktail to give target compound MalꢀLHRH. The purity of the MalꢀLHRH
peptide was confirmed using analytical HPLC and its identity was confirmed by mass spectrometry.
Platination with a 1.2ꢀfold excess of activated cisꢀ[Ptꢀ(NH3)2(H2O)2]2+(NO3)2 using a standard procedure
gave PtꢀMalꢀLHRH peptide shown in Figure 1. The platinum complexes could not be purified by HPLC
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using either an acidic or a neutral buffer system because the platinum complex dissociated. The products
were, therefore, purified by gel filtration to remove any excess platinum. Fractions containing the PtꢀMalꢀ
LHRH complex in water were identified by ESIꢀMS and lyophilized to PtꢀMalꢀLHRH give pure platinum
in a 15ꢀ17% yield. Mass spectra revealed the expected (M + Na)+ peak at 1756.7670 for the target
compound (see Supporting Information). Any experiment with PtꢀMalꢀLHRH had to use freshly
dissolved conjugate due to precipitates formed after storage, a phenomenon that has been reported before
by Ndinguri, et al.13 The PtꢀMalꢀLHRH compound was stable after the use of lyophilized powders at ꢀ20
°C. Before usage, fresh samples were made by reconstituting the dry samples to the desired
concentration.
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Mass spectrometry was used to verify the integrity of the redissolved PtꢀMalꢀLHRH. The final
complex yields a chemical structure similar to carboplatin, in which the carboplatin bioactive
region/structure is maintained (Figure 1).
Pt-Mal-LHRH increases breast cancer cytotoxicity through increased cellular uptake.
To examine the effects of PtꢀMalꢀLHRH on cell viability, 4T1 breast cancer cells were treated with
carboplatin, LHRH, or PtꢀMalꢀLHRH from 0.1 ꢁM to 100 ꢁM. PtꢀMalꢀLHRH was found to significantly
inhibit 4T1 viability compared to the untargeted carboplatin, concentrationꢀdependently (Figure 2A). Both
untargeted carboplatin and PtꢀMalꢀLHRH at 100 ꢁM was found to significantly attenuate cell viability
compared to the untreated control, however, this effect was more pronounced with PtꢀMalꢀLHRH. In
addition, the enhanced potency of PtꢀMalꢀLHRH was also verified using a second cell line, the MDAꢀMBꢀ
231 breast cancer cell line. PtꢀMalꢀLHRH was found to significantly attenuate MDAꢀMBꢀ231 breast cancer
cell viability compared to carboplatin (see Supporting Information). Moreover, the induction of cytotoxicity
by PtꢀMalꢀLHRH was confirmed by PI staining in which PtꢀMalꢀLHRH was found to significantly increase
apoptosis compared to carboplatin (Figure 2B). Numerous experimental and clinical studies have shown
overexpression of the LHRH receptor on human breast carcinomas, and specifically in the 4T1 and MDAꢀ
MBꢀ231 cell lines.28ꢀ29 To show the cytotoxic effect is induced by cellular entry of PtꢀMalꢀLHRH through
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binding to the LHRH receptor a competition assay was conducted. Treatment with free LHRH antibody
showed a significant attenuation in the ability PtꢀMalꢀLHRH to induce a cytotoxic effect (Figure 2C).
Subsequently, due to targeting LHRH receptor overexpression, PtꢀMalꢀLHRH cellular uptake was found to
be 20 fold higher than carboplatin in the 4T1 cell line (Figure 2D). PtꢀMalꢀLHRH uptake was also found to
be significantly increased in the MDAꢀMBꢀ231 breast cancer cells (see Supporting Information). These
results suggest that PtꢀMalꢀLHRH is more potent and efficacious than carboplatin in mediating breast
cancer cytotoxicity due to targeting aided by cellular overexpression of the LHRH receptor.
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Figure 2: Pt-Mal-LHRH attenuates breast cancer cell viability through increased cellular uptake.
4TI breast cancer cells were treated with PtꢀMalꢀLHRH, LHRH, or carboplatin (carbo) from a range of
0.1 ꢁM to 100 ꢁM for 24 hours. Viability rates were analyzed by a MTT assay after the cells were
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incubated for 48 hours (A). PtꢀMalꢀLHRH was Flow cytometric analysis of cell apoptosis was conducted
by treating cells with PtꢀMalꢀLHRH or carboplatin (100 ꢁM) for 4 hours, then washing the collected cells
followed by incubating the cells in 20 mg/ml PI for 15 minutes (B). Free LHRH antibody decreases the
cytotoxic effect of PtꢀMalꢀLHRH. Cells were pretreated with free LHRH antibody (100 ꢁM) for 30
minutes and then concurrently with PtꢀMalꢀLHRH (100 ꢁM) for 24 hours. Cellular viability was
analyzed by MTT assay after 48 hours (C). To measure drug uptake 1x106 4T1 cells were treated with
PtꢀMalꢀLHRH or carboplatin (100 ꢁM) for 24 hours. Cells were collected and metal (platinum)
concentration mg/L was measured by ICPꢀMS (D). (n=3); * p<0.05; ** p<0.01; *** p<0.001; ****
p<0.0001; twoꢀway ANOVA, oneꢀway ANOVA and Tꢀtest.
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Enhanced breast cancer cell selectivity is mediated by Pt-Mal-LHRH.
To determine the selectivity of PtꢀMalꢀLHRH towards breast cancer compared to normal fibroblast
cells, a cell viability assay was conducted. PtꢀMalꢀLHRH was found to significantly attenuate cell viability
in 4T1 cells compared to 3T3 normal fibroblast cells. This data indicates that the targeting ability of PtꢀMalꢀ
LHRH mediates selectivity towards breast cancer than normal cells (Figure 3).
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Figure 3: Pt-Mal-LHRH attenuates 4T1 breast cancer cell viability compared to normal cells (3T3).
4T1 and 3T3 cells were treated with PtꢀMalꢀLHRH from a range of 0.1 ꢁM to 100 ꢁM. Viability rates
were analyzed by a MTT assay after 72 hours of treatment. (n=3); **** p<0.0001; Twoꢀway ANOVA.
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Pt-Mal-LHRH decreases breast cancer cell migration in-vitro.
The foremost cause of mortality in breast cancer patients is metastasis, in which 10ꢀ15% of
women develop distant tissue colonization within 3 years after a primary tumor is diagnosed. 30 For this
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reason we conducted an in-vitro scratch assay to investigate the ability of PtꢀMalꢀLHRH to retard cell
migration. We found that PtꢀMalꢀLHRH significantly attenuated gap closure compared to untargeted
carboplatin and LHRH (100 ꢁM) treatments (Figure 4).
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Figure 4: Breast cancer cell migration is decreased by Pt-Mal-LHRH. Cultured 4T1 cells were scratched
under normal conditions (saline), carboplatin (carbo), LHRH, or Pt-Mal-LHRH (100 μM). Representative
10x images indicate Pt-Mal-LHRH treatment attenuated breast cancer cell migration (A). Cell migration
was quantitatively evaluated by measuring the distance between the scratch edges after 6 and 24 hours
(B). (n=3); **** p<0.0001; One-way ANOVA. Images were adjusted for brightness and contrast.
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Pt-Mal-LHRH attenuates breast cancer tumor growth and lung metastasis in-vivo.
To support our in-vitro results, we examined the antitumor activity and the effects on body and
liver weights of PtꢀMalꢀLHRH via in-vivo studies. Orthotopic 4T1 tumors grown in the right abdominal
mammary fat pad were treated with saline, carboplatin, carboplatin plus LHRH (unbound), or PtꢀMalꢀ
LHRH by intraperitoneal injection of molar equivalent doses for two weeks. We purposefully chose a low
treatment dose to optimally discern differences in the therapeutic effect of PtꢀMalꢀLHRH to carboplatin.
Previous publications highlight the use of higher treatment doses of carboplatin to mediate a significant
therapeutic effect on tumor growth.31ꢀ34 As predicted no significant decrease in tumor growth was found
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with carboplatin treatment compared to control mice; however, there was a significant decrease in tumor
volume with PtꢀMalꢀLHRH treatment compared to control tumors (Figure 5AꢀB). Additionally, no
difference in liver weights were found (Figure 5C) and cageꢀside observations did not detect any
noticeable side effects from PtꢀMalꢀLHRH treatment. Lung examination revealed a significant decrease in
lung tumor colonization in mice treated with PtꢀMalꢀLHRH compared to control and carboplatin treated
mice (Figure 5D). Subsequently, PtꢀMalꢀLHRH mice showed a slight decrease in lung weight compared
to control mice; however, significance was not reached (Figure 5E). Our in-vivo results indicate that Ptꢀ
MalꢀLHRH results in regression of the breast cancer volume and metastasis.
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Figure 5: Breast cancer tumor growth is attenuated by Platinum-LHRH treatment. Female
BALB/c mice were implanted with 4T1 cells (1x106) in the right second mammary fat pad. Tumors were
grown for 7 days, distributed into treatment groups, and treated with carboplatin (carbo), carboplatin and
LHRH, or PtꢀMalꢀLHRH (5mg/kg/wk) by intraperitoneal injection for 2 wks. Tumor volume was
measured over the 2 wk injection period (A) along with end tumor weight (B). Lung weight (C) Lung
tumor nodule formation (D) and liver weight (E) was measured. (n=7); * p<0.05; ** p<0.01; ****
p<0.0001; Two and Oneꢀway ANOVA.
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Taken together, synthesis of PtꢀMalꢀLHRH produced a targeting chemotherapeutic agent which is
more potent and selective towards breast cancer cells than carboplatin shown by our in-vitro results.
Subsequently, our in-vivo results suggest PtꢀMalꢀLHRH attenuates breast cancer tumor growth and
metastasis compared to control and carboplatin.
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Discussion
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The platinum family anticancer drugs are commonly used for chemotherapy of advanced
carcinomas. Our research is to develop the platinum drugs into targeting agents, in order to increase the
potency and efficacy while reducing systemic side effects. We synthesized the targeting
chemotherapeutic agent PtꢀMalꢀLHRH to target the overexpression of the LHRH receptor on cancer cells
relative to normal tissue. Studies have shown that LHRH receptors are overexpressed in breast, prostate,
endometrial and ovarian cancers in comparison to normal cells making the LHRH receptor a good
candidate for drug targeting.14ꢀ17 Specifically, numerous experimental and clinical studies have shown
overexpression of the LHRH receptor on approximately 50% of human breast carcinomas 35 and
specifically in the 4T1 cell line.28ꢀ29 Additionally, various nonꢀreproductive cancers have been shown to
overexpress the LHRH receptor including lung, bladder, pancreatic, among others.36ꢀ38
To form PtꢀMalꢀLHRH we have combined the two moieties (LHRH and activated cisplatin) using
a malonate linker making the new conjugate target specific. In our synthesized platinum conjugate, we
introduce an ester bond between the malonate linker and platinum. Prior published reports have shown
that such a bond is hydrolyzed by cellular esterases, leaving the drug free to act at the cellular level.12, 39ꢀ40
In our study we chose carboplatin as our untargeted platinum complex due to the fact that the malonate
linker chelates platinum in a manner similar to that of carboplatin, as has been previously reported.13 We
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have conjugated activated cisplatin using the malonate linker on position 6 to ensure that the intrinsic
properties of the LHRH peptide are maintained which would allow the delivery of the cytotoxic aquated
platinum to the tumor cells.
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One would expect that binding a platinumꢀbased chemotherapeutic compound with a targeting
agent should induce greater tumor toxicity with a concomitant reduction in systemic side effects.
Chemotherapeutic agents which are given systemically without a significant targeting advantage should
ideally have a low systemic toxicity or administered at a reduced dose to avoid complications. An
example of this would be the use of BEL for the treatment of breast tumors, which in the animal model,
has shown statistically significant reductions of tissue growth in both nicotine stimulated (p< 0.01) and
nonꢀnicotine stimulated breast cancer implants (p < 0.05).41 We hypothesize an increase in the
concentration of the drug PtꢀMalꢀLHRH in tumor cells as demonstrated in our in-vitro results would spare
the normal cells from unnecessary exposure and advantageously allow for a higher dose of PtꢀMalꢀLHRH
to be administered.
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An examination of the current literature involving the use of LHRH with platinum drugs as a
targeted treatment mechanism yields few results constituting effective or applicable targeted delivery. The
few published studies utilizing platinum derivatives employ combinatory methods on cisplatin resistant
tumors with LHRH and differ significantly in architecture and mode of delivery from the PtꢀMalꢀLHRH
complex reported in this research.42ꢀ43 For instance, LHRHꢀtargeted nanogels show complications with
free drug loading and slow release of the encapsulated cisplatin resulting in diminished cytotoxicity
compared to free cisplatin.43ꢀ44 However, our data demonstrated the successful synthesis of PtꢀMalꢀLHRH
and its effectiveness on breast cancer. Additional research examining the effectiveness of PtꢀMalꢀLHRH
on other cancers that express the LHRH receptor including but not limited to lung, bladder, and pancreatic
will need to be performed.
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Other modes of targeting platinum drugs that have been explored yielding limited results.
Targeted platinum sugar complexes have been shown to have differences in activities between D and L ꢀ
glucose conjugates; however, no increase in activity over the analogous carboplatin was found in either of
the complexes.45ꢀ47 Other complexes formed from a range of different sugars have been prepared, but the
results of the biological testing have not been released.47ꢀ48 Other studies targeting strategies for drug
delivery vehicles against the estrogen receptor (ER) in breast cancer have included steroids as targeting
units; however, these compounds did not fare better than cisplatin.46, 49ꢀ52 In one study, the platinum
conjugates cisꢀdichloro[Nꢀ(4ꢀ(17ꢀethynylestradiolyl)ꢀbenzyl)ꢀethylenediamine]platinum(II) and cisꢀ
diamino[2ꢀ(4ꢀ(17ꢀethynylestradiolyl)ꢀbenzoylamino)ꢀmalonato]platinumꢀ(II) enhanced proliferation
which made things worse and rendered them unsuitable for further investigation.53 It has been shown that
various cancer cell lines overexpress a glycoprotein that acts as a folate receptor. Cisplatin and
carboplatin derivatives bearing folate units were prepared; however, the conjugates had low solubility in
water which prohibited their use in biological experiments.46, 54 Aronov et al. used a PEG spacer to
enhance water solubility but the conjugate was found to be less cytotoxic than carboplatin.55 In regards to
peptideꢀbased targeting, instances in which targeting has been successfully achieved with platinum(II)
conjugates are few. One successful example is the use of cyclic peptide CNGRC sequence that targets the
CD13 receptor overexpressed on the surface of certain cancer cells.13
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The cytotoxic mechanism of action of PtꢀMalꢀLHRH mediates enhanced selectivity and efficacy
through use of the LHRH moiety to bind to the LHRH receptors which leads to internalization of the
compound. Our in-vitro data utilizing 4T1 cells supports the theory that PtꢀMalꢀLHRH increases
cytotoxicity through enhanced cellular uptake by over 20ꢀ fold increasing the bioavailability of
carboplatin and further augmenting its in-vivo tumoricidal properties. The increased tumor concentration
may also allow a decrease in the systemic dosage of the chemotherapeutic agent with a concomitant
decrease in system toxicity and side effects. This is an important consideration because of the
carboplatin’s narrow therapeutic index. Moreover, there are three cellular mechanisms which may impart
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a resistance to platinum based drugs. These include decreased uptake, increased repair of DNA damage
and increased drug deactivation.56 It would be expected that the enhanced uptake of platinum with the Ptꢀ
MalꢀLHRH compound may result in mitigating these resistance mechanisms, either directly or if
saturation can be obtained. Subsequently, once, PtꢀMalꢀLHRH is internalized it will be cleaved releasing
the aquated platinum to bind to the DNA forming DNA adducts resulting in cytotoxicity. We postulate
the DNA adduct formation will be similar to carboplatin which preferentially attacks the sequences of
GG>>AG>GA>GXG (X=an undefined residue), in that order.57ꢀ58
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One possible effect of PtꢀMalꢀLHRH is the targeting of the ovaries, adrenal and pituitary gland.
Chemical oophorectomy, adrenalectomy and hypophysectomy have been used in the treatment of
advanced breast cancer.59 If targeting of these organs take place, it is possible that it will be selective and
possibly affect only some of the endocrine functions. Interestingly, one study demonstrates that the
damaging effect to the pituitary gland by a targeted cytotoxic analog of LHRH was found to be reversible
after 2 wks of treatment.60 Further research into this area along with determining the exact mechanisms of
tumorcidal activity, and potential risks and benefits is needed.
In addition, we observed that LHRH treatment modestly stimulated tumor growth and migration.
This finding is expected since it is known that LHRH antagonists are effective breast cancer treatments.61
It has been shown that LHRH treatment promotes proliferation through reducing the number of resting
cells in the G0ꢀphase.62 Subsequently, our results demonstrate that tumor growth and metastasis
stimulated by LHRH is slightly greater than the control. However, we were unable to demonstrate tumor
cytotoxicity augmentation with the administration of unꢀlinked carboplatin with LHRH treatment;
therefore, the optimal timing, dosage and frequency of administration was not investigated.
We found PtꢀMalꢀLHRH significantly enhanced cellular cytotoxicity in 4T1 cells compared to the
3T3 normal cell line. Taken together, both our in-vitro and in-vivo data suggest PtꢀMalꢀLHRH elicits
tumorꢀtargeted drug delivery with increased potency, efficacy, and a possible reduction in
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chemotherapeutic side effects which would allow a full or higher dose of chemotherapy to be used in a
patient, as compared to other platinum drugs. Subsequently, our in-vitro scratch assay data demonstrated
a reduction in migration. Importantly, in-vivo metastasis was investigated since the major cause of
mortality in breast cancer patients is metastasis to distant sites including the lungs. Our in-vivo data
supports our cellular data, as we found a significant decrease in tumor volume and lung tumor
colonization through PtꢀMalꢀLHRH treatment.
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The advantage of the PtꢀMalꢀLHRH conjugate is selectivity toward tumors that overexpress
LHRH receptors locally at the site of their growth, while avoiding systemic distribution. Our approach to
tumorꢀtargeted drug/delivery will not be limited specifically to breast cancer, but could, in general, have
applications in other metastatic cancers that overexpress the LHRH receptor such as bladder, melanoma,
and pancreatic cancers, among others.
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In summary, we have demonstrated the synthesis of a new chemotherapeutic agent, PtꢀMalꢀ
LHRH, designed to selectively target cancer cells overexpressing the LHRH receptor. Our in-vitro results
using 4T1 breast cancer cells indicate increased cytotoxicity and uptake compared to carboplatin, along
with selectivity towards breast cancer compared to normal cells. In addition, our in-vivo results indicate
PtꢀMalꢀLHRH treatment significantly decreases tumor volume and metastasis to the lung. Our results
indicate that PtꢀMalꢀLHRH has increased potency, efficacy, and selectivity towards breast cancer cells
overexpressing the LHRH receptor compared to carboplatin. PtꢀMalꢀLHRH has the potential to be a
clinically used chemotherapeutic agent.
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Reagents
All starting reagents listed below were obtained from commercial sources and used without further
purification: N,Nꢀdiisopropylethylamine (DIEA), acetic acid, dichloromethane (CH2Cl2), trifluoroacetic
acid (TFA), Nꢀmethylmorpholine (NMM), triisopropylsilane (Tips), dry solvents, AgNO3, cisꢀ
diammineplatinum(II) dichloride (Cisplatin), were obtained from Fisher Scientific or Sigma Aldrich; all
amino acids and activators were from Novabiochem or AGTC Bioproducts.
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NMR Spectroscopy
The NMR spectroscopy was used to analyze and confirm the products obtained in each steps of
linker formation. All 1H NMR spectra were recorded on 400 MHz JEOL Eclipse+ NMR Spectrometer
and processed with DeltaTM NMR software.
Mass spectrometry
Analysis was done on a Thermo Scientific LTQ XL Mass Spectrometer using a DART (Direct
Analysis in Real Time) or (Electrospray Ionization) ESI on an Agilent Technologies instrument processed
with Analyst QS1.1 (Applied Biosystems) or Mass Hunter (Agilent)
HPLC
Analytical high performance liquid chromatography (HPLC) was performed to confirm purity of
LHRHꢀMal using an Agilent 1100 series multisolvent delivery system with a DAD detector controlled by
Agilent Chem Station Plus with detection at 220 nm.
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Inductive Coupled PlasmaꢀOptical Emission Spectroscopy
Platinum uptake was analyzed using a Varian, Vista MPX CCD simultaneous spectrometer.
PlasmaCal Pt (1000 µg/mL) from SCP Science was used for calibration by diluting it to standard
solutions of known concentration.
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Synthesis of Malonate (Mal) Linker
The malonate linker, Diꢀtertꢀbutyl 2ꢀ(3ꢀ(4ꢀCarboxybutanamido)propyl)ꢀmalonate (Mal) was synthesized
as previously described by Ndinguri et al.13 All analyses matched the reported 1H NMR and mass spectra
values.13
Synthesis of Malonate- LHRH (Mal-LHRH)
The MalꢀLHRH decapeptide was synthesized using Fmoc solid phase chemistry techniques and
then chelated to activated cisplatin. Briefly, FmocꢀRink AmideꢀAM resin (0.4 mmol) was placed onto a
reaction vessel. The resin was then washed with DMF and DCM in continuousꢀflow mode using a PS3
peptide synthesizer. All couplings employed four equivalents of amino acid and (2ꢀ(6ꢀChloroꢀ1Hꢀ
benzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethylaminium hexafluorophosphate (HCTU) and sometimes (7ꢀ
Azabenzotriazolꢀ1ꢀyloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyAOP) dissolved in 0.4 M Nꢀ
methylmorpholine (NMM) in DMF at room temperature. Coupling involved minimal preactivation times.
The side chainꢀprotected amino acid derivatives FmocꢀGlyꢀOH, FmocꢀProꢀOH, FmocꢀArg(Pbf)ꢀOH,
FmocꢀLeuꢀOH, FmocꢀDLys(Alloc)ꢀOH, FmocꢀTyr(tBu)ꢀOH, FmocꢀSer(tBu)ꢀOH, FmocꢀTrp(Boc)ꢀOH,
FmocꢀHis(Trt)ꢀOH and BocꢀGlu(OtBu)ꢀOH were used. The Alloc protecting group was removed using
tetrakis(triphenylphosphine)palladium(0) along with a 37:2:1 mixture of methylene chloride, acetic acid,
and NMM for 2 hours, followed by washing and double coupling of malonate linker (Mal).
Intermediate products were washed between reactions with DMF. The Fmoc group was deprotected with
20% piperidine in DMF for 3 min. Using a cocktail of trifluoroacetic acid: water: triisopropylsilane
(TFA:H2O:TIPS) (90:5:5:) (15 mL), the peptide was cleaved from the resin, precipitated and dried under
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vacuum to give the crude peptide conjugate MalꢀLHRH. MalꢀLHRH was purified by HPLC or gel
filtration using Sephadex Gꢀ10. The purified samples were lyophilized before use in the next step. The
crude peptide was purified using HPLC on reverse phase C18 column with a linear gradient from 5% to
80% B eluent in 15 min; tR 5.5 min (see Supporting Information). The purity of the compound was also
confirmed using mass spectrometry. Yield, 39%, ESIꢀMS (M+H)+, calculated for C70H101N19O20, 1528.69
found 1528.7304 (see Supporting Information).
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Synthesis of Pt-Mal-LHRH
Reaction of MalꢀLHRH and activated cisplatin yielded PtꢀMalꢀLHRH, see Figure 6. MalꢀLHRH
was dissolved in water, and the pH of the solution was adjusted to 7 by titration with 1 M NaOH. A
solution of cisꢀ[Ptꢀ(NH3)2(H2O)2]2+(NO3)2 was made by stirring cisꢀPtCl2(NH3)2 and AgNO3 vigorously
overnight in water in the dark. A precipitate of AgCl formed was filtered to give cisꢀ[Ptꢀ
(NH3)2(H2O)2]2+(NO3)2 (35, 36). A 1.2 fold excess of cisꢀ[Ptꢀ(NH3)2(H2O)2]2+(NO3)2 (20 mmol) was
reacted with MalꢀLHRH (30 mg, 20 mmol) to form the PtꢀMalꢀLHRH. Yield, 15ꢀ17%, ESIꢀMS (TOF)
gave a signal at 1756.760 (M + Na)+ calculated for C70H101N21O19Pt 1756.72
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Figure 6: Synthesis of Pt-Mal-LHRH. Synthesis of Mal-LHRH decapeptide using solid phase chemistry
and conjugation to activated cisplatin - Aminomethyl-ChemMatrix resin was coupled with 10 different
amino acids in the sequence indicated above. The allyloxycarbonyl (Alloc) protecting group on the D-lys
was selectively removed using palladium. The amine end of the D-lys was coupled with Mal and the
conjugate cleaved from the resin using TFA-Tips-H2O cocktail to yield LHRH-Mal. Reaction of LHRH-Mal
and activated cisplatin yields Pt-Mal-LHRH
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The 4T1 mouse mammary tumor cell line and 3T3 mouse embryonic fibroblast cell line was
obtained from ATCC and maintained as previously described.41 In addition, the MDAꢀMBꢀ231 cell line
was provided by Dr. Michael Kilgore at the University of Kentucky (purchased from ATCC which
authenticates the cell line through short tandem repeat (STR) profiling). The cells were maintained in
Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum and 100 U/ml
penicillin and 100 ꢁg/ml streptomycin.
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Cell Viability Assay
To assess the ability of PtꢀMalꢀLHRH to affect cell viability an MTT Assay was utilized. 4T1 or
MDAꢀMBꢀ231 cells were seeded 2000 cells/100µl in a 96ꢀwell plate. The wells were treated with PBS,
carboplatin, LHRH, or PtꢀMalꢀLHRH from a range of 0.1 ꢁM to 100 ꢁM for 24 hours. Cells were then
washed with PBS and cultured in DMEM supplemented with 10% FBS for 48 hours. After this the cells
were incubated at 37οC for 4 hours in 10 µl MTT solution obtained from Vybrant MTT Cell Proliferation
Assay Kit (Life Technologies). Cells were solubilized and mixed with SDS (sodium dodecyl sulfate) and
the absorbance read at 595 nm on a Phenix Genios Tecon 96 well plate reader.
Cytotoxicity Assay
Apoptotic cells were detected by flow cytometry using propidium Iodide (PI) staining of DNA
fragments to determine drug cytotoxicity. 4T1 cells were seeded 1X106 cells/well and treated with 100
ꢁM concentration of PtꢀMalꢀLHRH or carboplatin for 4 hours. Cells were collected, washed 3 times with
PBS and incubated with 20 µg/ml PI for 15 minutes. Next, flow cytometry was carried out using a
FACSCaliburTM flow cytometer (BectonꢀDickinson) at the University of Kentucky flow cytometry and
cell sorting core facility. A total of 50,000 events were acquired and analyzed.
Competition Assay
To determine binding of PtꢀMalꢀLHRH to the LHRH receptor for entry into the cell a competition
assay was conducted. 4T1 cells were seeded 2000 cells/100µl in a 96ꢀwell plate. Cells were pretreated
with free LHRH antibody (EMD Millipore Corp. AB1567) 100 ꢁM for 30 minutes and then concurrently
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with PtꢀMalꢀLHRH 100 ꢁM for 24 hours. Cells were then washed with PBS and cultured in DMEM
supplemented with 10% FBS for 48 hours. A cell viability assay was then performed as described above.
Drug Uptake Assay
To determine cellular uptake of PtꢀMalꢀLHRH, 4T1 cells were seeded 1X106 in 6 well plates and
treated with either carboplatin (100 ꢁM) or PtꢀMalꢀLHRH (100 ꢁM) for 24 hours. Cells were washed 3
times with PBS, collected, and metal (platinum) concentration mg/L as measured by ICPꢀMass
spectrometry at Louisiana State University as previously described.13
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Cell Migration
Cell migration was examined using a scratch assay. 4T1 cells were cultured in a 6ꢀwell plate
until a confluent monolayer was formed. The wells were scratched with a 20 ꢁl pipet tip, rinsed with PBS,
and cultured in DMEM supplemented with 10% FBS. The wells were treated with PBS, carboplatin,
LHRH, or PtꢀMalꢀLHRH. A concentration of 100 ꢁM was used for all compounds. Four representative
10x images were taken at 0 and 24 hours and the gap width was quantified using an average of three
leading edge measurements for each image.
In-vivo Syngeneic Isograft Model of Breast Cancer
Female BALB/c mice, 10 weeks old with an average weight of 19.80g, were purchased from
Jackson Laboratory (Bar Harbor, ME) and housed as previously described.41 Animal protocols were
approved by the committee on animal research care and use at Eastern Kentucky University.
4T1 cells (1X106) were suspended in 100 µl of DMEM not supplemented with FBS and injected
into the right second mammary fat pad of female BALB/c mice as previously described in literature.63
After 7 days of orthotopic tumor initiation (~100mm3) the mice were treated with saline, carboplatin,
carboplatin plus LHRH, or PtꢀMalꢀLHRH by intraperitoneal injection for two weeks. A molar equivalent
dosage of 5mg/kg/wk was used for all compounds according to their molecular weights. Tumor growth
was monitored daily and tumor volumes (mm3) were calculated using the formula: (width)2 × length/2,
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where width is the smaller of the two measurements. After 2 weeks of treatments the mice were sacrificed
and tumor volume and organ weights were measured.
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Statistics
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Data are illustrated as mean ± SEM and statistical analyses were carried out using GraphPad,
Prism 6 (San Diego, CA) using tꢀtests. Oneꢀ or twoꢀway ANOVAs were used where appropriate.
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Acknowledgements: We would like to thank Dr. Thomas Blanchard for his help with the ICP analysis.
Funding Sources: Kentucky Biomedical Research Infrastructure Network (KBRIN) grants
(P20GMI04489 and P20GM103436); EKU University Research Grant and National Institute of General
Medical Sciences at the National Institutes of Health (grant # 1R25GM102776ꢀ01)
Supporting information: Supporting Information Available: MDAꢀMBꢀ231 cytotoxicity graph,
MDAꢀMBꢀ231 uptake graph, HPLC analysis spectra and mass spectra analysis.
This material is available free of charge via the Internet at http://pubs.acs.org
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Abbreviations: N,Nꢀdiisopropylethylamine (DIEA), dichloromethane (CH2Cl2), trifluoroacetic acid
(TFA), Nꢀmethylmorpholine (NMM), triisopropylsilane (Tips), Dimethylformamide (DMF), cisꢀ
diammineplatinum(II) dichloride (Cisplatin), 2ꢀ(6ꢀChloroꢀ1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀ
tetramethylaminium hexafluorophosphate (HCTU), 7ꢀAzabenzotriazolꢀ1ꢀ
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yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyAOP), Dulbecco’s Modified Eagle Medium
(DMEM), 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT), American Type Culture
Collection (ATCC), Fetal Bovine Serum (FBS)
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