|Home | About | Journals | Submit | Contact Us | Français|
Clinical experiences often document, that a successful tumor control requires high doses of drug applications. It is widely believed that unavoidable adverse reactions could be minimized by using gene-therapeutic strategies protecting the tumor-surrounding healthy tissue as well as the bone-marrow. One new approach in this direction is the use of “Targeted Therapies” realizing a selective drug targeting to gain effectual amounts at the target site, even with drastically reduced application doses. MCF-7 breast cancer cells expressing the αvβ3 [alpha(v)beta(3)] integrin receptor are considered as appropriate candidates for such a targeted therapy. The modularly composed BioShuttle carrier consisting of different units designed to facilitate the passage across the cell membranes and for subcellular addressing of diagnostic and/or therapeutic molecules could be considered as an eligible delivery platform. Here we used the cyclic RGD-BioShuttle as a carrier for temozolomide (TMZ) at the αvβ3 integrin receptor realizing local TMZ concentrations sufficient for cell killing. The IC50 values are 12 µMol/L in the case of cRGD-BioShuttle-TMZ and 100 µMol/L for underivatized TMZ, which confirms the advantage of TMZ reformulation to realize local concentrations sufficient for cell killing.
Our paper focuses on the design, synthesis and application of the cRGD-BioShuttle conjugate composed of the cyclic RGD, a αvβ3 integrin-ligand, ligated to the cytotoxic drug TMZ. The ligation was carried out by the Diels Alder Reaction with inverse electron demand (DARinv).
Breast cancer is one of the most common malignancies affecting women in developed countries.1 Approximately three out of four women with breast cancer develop metastases in bone which, in turn, diminish the quality of life.2 An optimal treatment concept for patients needs different therapy modalities and methods with an optimum in efficiency and the greatest possible protection. Attention should be laid on an individual and not just standardized plan of treatment for every single patient and all available therapy options should be used, such as immunotherapy, surgery or chemotherapy sensibly using cytostatic active agents with acceptable adverse reactions. It is remarkable how dated medical treatment methods are persistently continued [reported during the “International Brain Tumor Research Conference 2010 (http://www.kgu.de/index.php?id=4290)].
Toxic side effects are documented for TMZ as adverse reactions in the bone-marrow. Moreover, it is known from clinical experience, that even higher application doses are necessary for successful tumor control. This approach seems obsolete now, because 'Targeted Therapy' has reached the focus of scientific interest in order to minimize such unavoidable drastic side effects. Strategies were discussed during the aforementioned meeting to protect the bone-marrow, e.g. with gene-therapeutic methods. Another interesting field is the regional chemotherapy in which cytostatic drugs are being locally applied to certain body regions. The topical application increases the amount of active substances in the tumor and improves efficiency, while lowering the side effect rate at the same time.
However, many cell immanent obstacles inhibit chemical therapy, such as the multidrug resistance (MDR) mediated against cytotoxic agents like TMZ, and apoptosis resistance with disruption of the complex programmed cell death pathway network. The Janicke group documented apoptosis resistant MCF-7 breast cancer cells treated with ionizing radiation, however especially breast micro-metastases are difficult to determine and even more difficult to treat effectively.
Therefore only a selective targeting of the drug can deliver an effectual amount of TMZ to its target site, even with drastically reduced application doses. How to perform this is exemplarily shown here by targeting and controlling breast cancer cells.
Our considerations to overcome these resistance-inducing factors led to the application of ligands, which are target-specific for cell-typical surface receptors, as described as follows.
On these cells the αvβ3 [alpha(v)beta(3)] and αvβ5 [alpha(v)beta(5)]integrins are heterodimeric cell surface receptors which mediate adhesion between cells and the extracellular matrix.3 The αvβ3 receptor has previously been implicated in a key role of tumor progression, metastasis and osteoclast bone resorption. 4 Integrins, the corresponding ligands, are evolutionarily old and have critical roles during developmental and pathological processes. The antibodies to αvβ3 integrin and its antagonists like arg-gly-asp (RGD)-containing peptides, including osteopontin, bone sialoprotein, vitronectin and fibrinogen are considered as efficient inhibitors which can control the tumor progression.5
Endocytosis-mediated intracellular trafficking of ligands via the αvβ3 receptor of MCF-7 cells and the αvβ5 integrin receptor into the perinuclear region of HeLa cells is documented, which lack the functional αvβ3 receptor.10 Interestingely HeLa cells, which express the αvβ3 integrin receptor at low level, possess lower invasive potential than MCF-7 cells. In our experiments we used MCF-7 human breast cancer cells and HeLa cervix cancer cells to investigate the new cRGD-BioShuttle as a delivery platform for targeting with TMZ in order to realize high local TMZ concentrations at the MCF-7 and HeLa cell's surfaces and, after uptake into the cells sufficient for cell killing.
This paper intends to summarize the major efforts reached thus far and focuses on the design, synthesis and application of the cRGD-BioShuttle-TMZ conjugate. The whole molecule was synthesized via Diels Alder Reaction with inverse electron demand. It is composed of the cyclic RGD-containing the αvβ3 and αvβ5 integrin antagonist cRGD.
The estrogen sensitive MCF-7 adenocarcinoma breast cancer and HeLa cervix cancer cells (dkfz, tumorbank) were maintained at 37°C in a 5% CO2 atmosphere in RPMI cell medium (Gibco, Germany) supplemented with 5% fetal calf serum (Biochrome, Germany). The cells were split twice a week.
4-(6-(Pyrimidine-2-yl)-1,4-dihydro-1,2,4,5-tetrazine-3-yl)benzoic acid (3) was prepared from 2-cyanopyrimidine 1 and 4-cyano-benzoic acid 2 by reaction with hydrazine and then oxidized with sodium nitrite to the tetrazine derivative 4 according to the following procedure 11. The tetrazine derivative was converted with thionyl chloride under standard conditions to the chloride 5. To this suspension of the acid chloride (2 mmol) in 20 ml CH2Cl2 a solution of N-Boc-1,3-diaminopropane (2 mmol) and TEA (2 mmol) in 10 ml CH2Cl2 was slowly added at 0-5°C. The resulting solution was deeply coloured and maintained for 4 h at room temperature. Then the organic phase was washed with water, followed by 1N HCl and again water. The organic layer was dried over Na2SO4 and evaporated. The resulting residue was chromatographed on silica gel by elution with chloroform/ethanol (9:1) and further purified by recrystallization from acetone. Yield: 50 to 70 % depending on the quality of the carboxylic acid. ESI MS: m/z 437.2 [M]+. The Boc-protected derivative was treated with TFA (5 ml) for 30 min at room temperature and isolated by evaporation to a solid residue (6) (ESI: m/z 337.2 [M]+ (as shown in Figure FigureAA).
3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxylic acid was converted to the corresponding chloride 7 as documented by Arrowsmith 13. The acid (2 mmol) was refluxed with thionyl chloride (10 ml) until the acid was completely dissolved. The excess of thionyl chloride was evaporated under vacuum and the resulting solid was stored over NaOH.
Compound 8 (0.5 mmol) and the chloride 7 (0.5 mmol) were dissolved in 5 ml chloroform and 5 ml TEA at 0-5 °C. After 4 h at room temperature, the solution was washed with water, 1 N HCl and again with water. The organic layer was dried over Na2SO4 and evaporated. The residue was purified by chromatography (silica gel) with chloroform/ethanol (9.5/0.5). Yield: 68%: ESI: m/z 536.3 [M+Na]+. (Figure (FigureBB)
The tetracyclo-[5.4.21,7.02,6.08,11]3,5-dioxo-4-aza-9,12-tridecadiene (Reppe-anhydride) 12 was prepared from 42 mg of (1Z,3Z,5Z,7Z)-cycloocta-1,3,5,7-tetraene 10 and 44 mg maleic anhydride 11 in chloroform as documented by Reppe 14.
30 µmol cRGD peptide (18 mg) 13 and 40 µmol (8 mg) tetracyclo-[5.4.21,7.02,6.08,11]3,5-dioxo-4-aza-9,12-tridecadiene 12 were dissolved in pyridine over 5 hours at 70° - 80°C. Yield: 6 mg 14. Empirical formula C39H49N9O9; exact Mass: 787.37 Mol. Wt.: 787.86
m/e: 787,37 (100,0%), 788,37 (43,1%), 789,37 (12,3%), 788,36 (3,3%), 790,38 (1,2%), 790,37 (1,1%) C, 59.45; H, 6.27; N, 16.00; O, 18.28 m/e peak at 788.5 for the product.
Equimolar amounts of the TMZ-tetrazine conjugate 9 (1.03 mg; 2 µmol) and cRGD-Lys(Tct) 14 (7.3 mg, 2 µmol) were dissolved in aqueous solution and stored at room temperature for 24 h. The DARinv reaction occurs at room temperature and was completed after the colour changed from magenta to yellow. The product 15 (cRGD-BioShuttle-TMZ) was isolated by lyophilization; yield: 98 %); MS ESI: m/e 1634.8; calculated C81H91N19O15S2 1633.6.
For investigations of the cellular localization of the cRGD we functionalized the cRGD with the fluorescent dye 5-(dimethylamino)-naphthalene-1-sulfonyl, (dansyl, as shown in scheme 4/Figure 4/FigureS4S4).
2 µmol (1.75 mg) of purified 14 reacts over 24 hours with 2.5 µmol (2.18 mg) 16 dissolved in DMSO at room temperature to the product 17. The reaction mixture was concentrated. After HPLC purification the estimations of identity show in position 1634, Mode m/e.
Exact Mass: 1633.64; Mol. Wt.: 1634.84
m/e: 1633.64 (100.0%), 1634.64 (97.8%), 1635.65 (39.3%), 1636.65 (14,2%), 1636.64 (11.9%), 1635.64 (10.8%), 1635.63 (9.4%), 1637.65 (4,7%), 1637.64 (4,2%), 1638.64 (1.6%)
C, 59.51; H, 5.61; N, 16.28; O, 14.68; S, 3.92
Pure temozolomide (TMZ) [Sigma-Aldrich, Germany (Cat. No. 76899)] was subdivided into two parts for subsequent processing. One part was kept underivatized for the following experiment and the second part, after chemical transformation to the corresponding acid chloride 7, was used for coupling to the cRGD [Peptides International, USA (Cat. No. PCI-3661-PI)] transporter molecule.
TMZ and cRGD-BioShuttle-TMZ 15 were both dissolved in 10 % aqueous solution of acetonitrile (Sigma-Aldrich, Germany). Control studies with acetonitrile were performed to exclude potential toxic effects of this solvent.
MCF-7 and HeLa cells were grown as subconfluent monolayers in RPMI (control) and in RPMI containing appropriate amounts of TMZ and the cRGD-BioShuttle-TMZ 15 (50 µM) and their behaviour was analyzed for up to 72 hours.
Microscopical studies of the human cancer cells were carried out with an Olympus inverted microscope under phase contrast conditions. The magnification was 200fold. The cells were observed during their culture in medium and during treatment with the different drugs.
In order to reconfirm our data documenting an endocytotic internalization of the cRGD into the cytoplasm of αvβ3 and αvβ5 integrin expressing cells we used the confocal laser scanning microscope (CLSM) of the Microscopy Core Facility of the German Cancer Research Center for qualified verification of the data. The pictures were taken with a Leica confocal microscope TCS SP5 II (excitation at 405 nm, emission at 420-560 nm) and examined with the Leica LAS-Software.
24 hours before CLSM measurements both cell lines, the HeLa and the MCF-7 cells (5×105), were cultivated in 8-well cell culture plates (Lab-Tek®) and treated with the cRGD-BioShuttle-dansyl (12.5 µM) 17.
For the toxicological characterization of the cRGD-BioShuttle-TMZ conjugate 15 as well as the cRGD and the TMZ alone as controls were added to the MCF-7 cell line's medium. The substances were incubated in a dilution series ranging from 12.5, via 25, 50, to 100 µM final concentrations up to 72 hours. The IC50 values were determined and also converted to the pIC50 scale (-log IC50) (Table (Table11).
The use of the flow cytometry parameters forward (FSC) and sideward (SSC) scatter of the cells give an indication on drug effects through the relative cell size and structural effects such as granularity. Both parameters suffice for a rough cell characterization. The cells (treated with the components as described above and an untreated control) could be clearly distinguished as shown in the Figure Figure55.
This manuscript details the synthetic steps of our new cRGD-BioShuttle-TMZ and illustrates the cellular uptake of this newly synthesized cRGD-BioShuttle-TMZ in comparison to its controls as outlined in the respective experiments. We examined MCF-7 and HeLa cells surface targeting of these molecules with the cytotoxic drug TMZ as a cargo.
In light microscopy we first investigated the cell killing effect of cRGD-Bioshuttle-TMZ 15 compared to underivatized TMZ. We achieved a rapid and high local concentration and an accumulation of TMZ on the surface of the targeted αvβ3 integrin expressing MCF-7 cells by use of the cRGD-BioShuttle as delivery and targeting platform.
Figure Figure11 reveals the different effects of TMZ and cRGD-BioShuttle-TMZ on MCF-7 cells tested after 24 hours and 72 hours of treatment with a final concentration of 50 µM. Whereas the MCF-7 cells exhibit no formation of the squamous epithelium (B), the MCF-7 cells seem to be unimpressed by TMZ treatment (bottom row C) and resemble the untreated control (top row A) as shown in the microscopic pictures.
Figure Figure22 indicates a clear change of the MCF-7 phenotype dependent on the concentrations of cRGD-BioShuttle-TMZ of up to 50 µM. The untreated control cells are shown in the bottom row for each treatment regimen. The final concentrations of the cRGD-BioShuttle-TMZ and TMZ were from 12.5 µM, via 25 µM to 50 µM as indicated in the figure figure2.2. A drastic cell killing of the MCF-7 cells was observed by the targeted approach, whereas the MCF-7 cells treated with underivatized temozolomide seemed to be not affected. Independent of the final TMZ concentrations used, they looked identical to the untreated control cells.
In order to investigate the open question of the cellular localization of the cRGD-BioShuttle-TMZ we ligated a fluorescence dye to cRGD. This “BioShuttle” (cyclo RGD) connected to a fluorescent tag dansyl (as shown in scheme 4/Figure 4/FigureS4)S4) was applied to the culture media of the αvβ3 and αvβ5 integrin expressing MCF-7 and the low expressing HeLa cells. The cells lines clearly show differences in the fluorescence signal localizations. Two hours after cRGD-BioShuttle-dansyl application a blue perinuclear dansyl fluorescence signal could be observed in MCF-7. The cell nucleus displayed no signal at all. The HeLa cells show a cells surface located fluorescence signal (Figure (Figure3,3, top row).
24 hours after cRGD-BioShuttle-dansyl application the fluorescence signal increased in the cytoplasm of MCF-7 and at the surface of HeLa cells, but the signal localizations remained unaltered. A nuclear located fluorescence signal was still lacking (as shown in Figure Figure3,3, second row). The pictures from 48 and 72 hours after application demonstrate a decreased fluorescence signal suggesting an efflux of the dansyl fluorescence dye out of the MCF-7 cell's cytoplasm, whereas in contrast the fluorescence signal at the HeLa cell's surface is unchanged (Figure (Figure3,3, row 3 and bottom row).
For detailed information about the cellular localization of the dansyl fluorochrome 8 pictures of a z-stack were visualized in layers by CLSM. It is demonstrative, that some fluorescence signals are shown inside of the cells, but the signal is not only localized at the cell's surface but also detectable inside as pointed out in the legend of the figure figure44.
The morphological parameters of the FACS analysis shows an unaltered cell size (Figure (Figure5,5, bottom row), whereas the granularity (influenced by size and structure of the cell nucleus and by the quantity of vesicles) is changed and shows an increased fraction of more granulized MCF-7 cells in all treatments (TMZ, cRGD, and cRGD-BioShuttle-TMZ) in contrast to the untreated MCF-7 control cells. As demonstrated in figure figure55 the granularity of MCF-7 cells is increased in cells treated with TMZ and cRGD (100 µM respectively). The most conspicuous granularity was obtained after cRGD-BioShuttle-TMZ in the final concentration of 12.5 µM as shown in the blot of the figure figure55 (right column, row 1).
The treatment of TMZ, cRGD, and cRGD-BioShuttle-TMZ in the concentrations as mentioned above shows no visible influence on the cell size of MCF-7 cells.
A comparison of the flow cytometry data (Table (Table1)1) to the CLSM data as shown in Figure Figure33 right column is useful. In the image of the 24 hours time point after application with cRGD-BioShuttle-dansyl the MCF-7 cells show a clear perinuclear endoplasmic reticular located blue fluorescence signal which represents an increase in the granularity resulting from the unstained endoplasmic reticulum surrounding the nucleus. As demonstrated in the Figure Figure5,5, second row, the cells treated with 100 µM TMZ show an increased amount of granular cells (20 %) compared to the untreated control with 4.5% granularity. In the sample of MCF-7 cells treated with the cRGD alone (100 µM), a cellular granularity of 33.2 % was measured. It is important to note that the MCF-7 cells treated with cRGD-BioShuttle-TMZ in an at least 8-fold reduced application dose (12.5 µM) feature the highest cell response with a cellular granularity of 34.2%.
To measure the chemotherapeutic sensitivity against the investigated components, the TMZ, the cRGD, and the cRGD-BioShuttle-TMZ-conjugate were added to MCF-7 cells. Again, the substances were dissolved in an aqueous dilution series in culture medium in a concentration from 12.5 µM to 100 µM and applied over 72 hours. The data are presented in Table Table1.1. With the flow cytometry analyses we showed the high sensitivity of MCF-7 against cRGD-BioShuttle-TMZ with an IC50 value of 12.5 µM, and a much lesser sensitivity against underivatized TMZ with an IC50 of 100 µM). The MCF-7 cells treated with cRGD (12.5 up to 100 µM) were unimpressed.
As definitive treatments of metastases of breast cancer remain surgery, radiation therapy, and hormone therapy in the case of metastatic progress. Cytogenetic characteristics, like the range of the chromosome number between hypertriploidy and hypotetraploidy, could answer the question of the recalcitrance of metastatic breast cancer cells, exemplarily MCF-7 cells, against therapeutic interventions. But the timing of treatment for all stages of the disease remains controversial and new therapeutic approaches are needed 15, 16. Encouraging results of the chemotherapeutic TMZ treatment in brain tumors 17 remain unendorsed in the treatment of breast cancer cells.
A May 28th 2010 search in the NCBI database PubMed with “RGD” “MCF-7” yields 26, and with “RGD” “HeLa” 89 hits. In its first 1995 publication the Huang group described inhibiting features of RGD-containing peptides against αvβ3 integrin expressing cells 18 whereas the activity of αvβ5 integrin was already documented from the Boulanger and Nemerow groups in 1993 19, 20.
An explanation is that the integrin αvβ3 receptor is expressed at low levels in epithelial and mature endothelial cells but is upregulated on tumor cells and tumor endothelial cells of varying tumor types including breast cancer, and therefore became considered as a prognostic factor in breast cancer 21. It was also documented that peptide ligands containing RGD amino acid sequences have a high affinity for integrins and after labelling with radioisotopes, could be used for imaging αvβ3 receptor levels by PET or SPECT studies 22. The use of RGD peptides ligated with imaging components for cell specific targeting as tumor-diagnosing tool is broadly documented 23, 24. All these documented radioligands may be valuable for monitoring antiangiogenic therapeutics. The ligation of such therapeutically active substances to RGD peptides could open a door for successful treatment of hardly tractable tumors like metastases expressing αvβ3 integrin. In comparison with the normal expression level of MCF-7 mammary cancer cells, αvβ3 low expressing HeLa cervix cancer cells were examined, which express the αvβ5 integrin and correlate with low invasiveness.
In flow cytometry, the increased relative amount of cells revealing granularity and the accession of the cell fraction featuring a clear augmentation of the granularity both might result from increased numbers of affected mitochondria, golgi apparatus or endoplasmatic reticulum. This could be explained with a pharmacologic effect of the cRGD, as well as of the TMZ alone. The BioShuttle construct containing the cRGD as targeting modul connected to the TMZ via the Diels-Alder Reaction with inverse electron demand 12 targets the αvβ3 and αvβ5integrin MCF-7 cell surface proteins as shown in the Figure Figure3.3. According to CLSM-pictures of the MCF-7 cells (Figure (Figure3),3), an increased cytoplasmic coloring is indicative for a strongly increased amount of granularity in cells (R2) in the cytometric results. It is important to note that no changes in cell size could be observed.
The results of the present study demonstrate a close relationship of the reformulated drug to unanswered clinical questions as suitable solutions for the patients (as described in the introduction). Here we ligated TMZ to the cRGD, a peptide-based component, which accumulates at the cell's membrane surface holding a tremendous potential to optimize therapy. Enhanced local concentrations of active substances like TMZ at the cells surface, a feasible site of pharmacological action, allow expecting lower application doses with concomitantly decreased side-effects. The uptake and internalization into the cells cytoplasm is presumably documented 25. The criteria to determine the applicability for targeting including accessibility, specificity, safety and subcellular precision are documented 26-30. In order to fulfill all these biomedical aspects, the TMZ drug was bound as a cargo to transporter molecules (cRGD-BioShuttle-TMZ) with special chemical methods 31. These coupling reactions however, required the reformulation of the active drug TMZ and can lead to novel properties of the TMZ. Chemoselective reaction-conditions in aqueous solution and at room temperature were obtained by chemical ligation of functional peptides via the DARinv. The “click chemistry” based on the DARinv with “inverse electron demand” resulted in an impressive efficiency as illustrated in scheme 3/Figure 3/FigureS3S3 and is well documented 12. This reaction enhanced the economics of the chemical reaction by the following parameters: ① increase of the reaction rate, ② gentle reaction conditions at room temperature, and ③ resulting in reaction kinetics with stoichiometric reaction partners without excess of the educts. The BioShuttle-mediated delivery and targeting platform can facilitate the transport to target cells 31, 32. Despite the fact that the IC50 value can vary, depending on factors like the cell line, the test protocol, and the investigator, this estimation gives useful and helpful information on the relative toxicity 32. Regarding the difference in IC50 values between the TMZ at 100 µmol/L and cRGD-BioShuttle-TMZ at 12.5 µmol/L, it becomes obvious that the cRGD-BioShuttle-TMZ offers a high potential for treatment of patients and represents an attractive enhanced drug system for upcoming clinical combined chemotherapeutic approaches 33, 34.