10-Hydroxycamptothecin (HCPT) elicits strong anti-cancer effects and is less toxic than camptothecin (CPT), making it widely used in recent clinical trials. However, its low solubility limits its application as an effective anti-cancer therapy. In the present study we investigate the hypothesis that the unique water dispersible oleic acid-Triton X-100-coated Fe3O4 nanoparticles loaded with HCPT disrupt epithelial cell–cell junctions and induce human lung cancer cell apoptosis through the caspase-8 pathway. We characterized the HCPT-loaded nanoparticles and determined their effects on lung cancer cell viability and apoptosis by using immunofluorescence light microscopy and SDS-PAGE/immunoblots. We found that HCPT-loaded nanoparticles elicited an anti-proliferative effect in a dose-dependent manner. HCPT-loaded nanoparticles reduced the expression of cell–cell junction protein claudins, E-cadherin and ZO-1, and transmission electron microcopy demonstrated a disrupted tight junction ultrastructure. Transepithelial electric resistance was also reduced, indicating the reduction of tight junction functions. The HCPT-loaded nanoparticles increased phosphorylation of p38 and SAPK/JNK while it showed no effects on p42/44 MAP kinase. Compared with void Fe3O4 nanoparticles or HCPT drug alone, HCPT drug-loaded nanoparticles evoked synergistic effects by increasing cell apoptosis with enhanced activation of the caspase-8 pathway. Therefore, our current study highlights the potential of HCPT drug-loaded nanoparticles as a chemotherapeutic agent for increasing anti-cancer drug efficacy. (Cancer Sci 2011; 102: 1216–1222)
Camptothecin (CPT) is a natural alkaloid extracted from the Chinese tree, Camptotheca Acuminata Decne. Due to its potent anti-cancer activity, CPT and its derivatives have received increasing attention in recent years. The anti-tumor mechanism of these compounds is based on the inhibition of DNA replication and RNA transcription by stabilizing the cleavable complexes formed between topoisomerase I (topo I) and DNA. The complex formation prevents DNA re-ligation and causes DNA damage, which results in apoptosis.(1) Among its analogues, 10-hydroxycamptothecin (HCPT) shows strong anti-tumor effects and is less toxic compared with CPT. 10-Hydroxycamptothecin has been widely used in the treatment of a variety of cancers in clinical trials.(2) Unfortunately, HCPT has poor solubility in physiologically acceptable solvents and is usually used in the more soluble carboxylate salt form that is more toxic and less active.(2)
Nanotechnology offers promising applications in cancer treatments due to the unique properties of nanostructures. Drug-coated polymer nanoparticles could efficiently increase the intracellular accumulation of anti-cancer drugs.(3) One emerging application is to use surface modified Fe3O4 magnetic nanoparticles as vehicles for drug delivery.(4) However, relatively high toxicity of uncoated magnetic nanoparticles restricts their use in humans. Therefore, many approaches have focused on the encapsulation of magnetic nanoparticles with biocompatible materials to reduce toxicity.(5,6) Triton X-100, a derivative of polyethylene glycol (PEG), is widely used in biological applications due to its low toxicity and good biological compatibility.(7) Recently, Jain et al. reported a water-dispersible oleic acid (OA)-Pluronic-coated iron oxide nanoparticle formulation that can be loaded easily with high doses of water-insoluble anti-cancer agents.(4)
The nanocomposites of polylactide (PLA) nanofibers and tetraheptylammonium-capped Fe3O4 magnetic nanoparticles have been tested using a different anti-cancer drug daunorubicin,(3,8) which can inhibit tumor growth.(9) The nanoparticles composed of chitosan/poly-γ-glutamic acid could affect tight junction (TJ) function.(10) However, the feasibility of Fe3O4 magnetic nanoparticles loaded with HCPT in the presence of Triton X-100 has not been investigated. There are significant interests in revealing whether this nanoparticle-based technology coupled with HCPT can induce cancer cell death through well-characterized molecular and cellular signaling mechanisms.
To overcome these limitations, we have developed an efficient delivery system that uses Fe3O4 magnetic nanoparticles loaded with a high dose of HCPT in the presence of Triton X-100. We report that HCPT drug-loaded nanoparticles induce apoptosis in the human lung cancer cell line HCC827, as determined by molecular, biochemical and morphological methods. We find that nanoparticles and HCPT drug-loaded nanoparticles disrupt TJ and increase the permeability of epithelial monolayers. Furthermore, we demonstrate that the cancer cell inhibitory effects induced by HCPT drug-loaded nanoparticles involve activation of the caspase-8 as well as p38 and SAPK/JNK signaling pathways.
Materials and Methods
Preparation of magnetic Fe3O4 nanoparticles and drug-loaded nanoparticles. FeCl3 was suspended in dH2O under a nitrogen-gas atmosphere to remove oxygen from the mixture, followed by the addition of FeCl2 to the mixture. Ammonia solution was added drop wise with stirring until the mixture had pH 9.0–10.0. The particles obtained were washed with nitrogen-purged water. Oleic acid (OA) was added to the above solution of particles. The mixture was stirred at 80°C to evaporate the ammonia and then cooled to room temperature. Triton X-100 was added to the OA-coated nanoparticles, and the suspension was stirred overnight. 10-Hydroxycamptothecin dissolved in DMSO was added to the aqueous dispersion of OA-Triton X-100-stabilized Fe3O4 nanoparticles (TOFN). The mixture underwent stirring overnight in a nitrogen gas atmosphere to allow partitioning of the drug into the OA shell surrounding the Fe3O4 nanoparticles. Drug-loaded nanoparticles (HTOFN) were washed and separated from the unentrapped drug using a magnet.
Transmission electron microscopy. The HTOFN were observed under transmission electron microscopy (TEM) and the samples for analysis were prepared by applying the diluted suspensions onto carbon-coated copper grids and allowing the solvent to evaporate.
To investigate the effects of HTOFN on cell–cell junctions, HCC827 cells were incubated with HTOFN and then fixed in 2.5% glutaraldehyde. Ultrathin sections were viewed under a JEOL 1200-EX transmission electron microscope (JEOL Ltd., Tokyo, Japan) and images were taken using a MegaView III CCD camera (Olympus Soft Imaging Solutions, Munster, Germany) and ITEM imaging software (Olympus Soft Imaging Solutions, Munster, Germany).
X-ray diffraction. The HTOFN were characterized by X-ray powder diffraction (XRD) (Rigaku D/Max-IIIC, Tokyo, Japan) using Co K[alpha] radiation: 1.789 Å. Distances between peaks were compared with the information related for diffraction data to determine crystalline structures.
Cell culture and MTT assay. HCC827 (human non-small-cell lung cancer) cells from American Type Culture Collection (ATCC, Manassas, VA, USA) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA) at 37°C in a humidified air, 5% CO2 atmosphere.
For cytotoxicity analysis, MTT assays were performed on cells treated with various concentrations of HTOFN, void nanoparticles (TOFN) and HCPT (dissolved in DMSO with the final concentration 0.2%). The optical density (OD) was read at a wavelength of 540 nm. All experiments were performed in triplicate. Relative inhibition of cell growth was expressed as follows: % cell growth inhibition = (1 – [OD]test/[OD]control) × 100%.
Apoptosis measurements. Apoptotic DNA was extracted from the HCC827 cells using an Apoptotic DNA ladder Isolation kit (BioVision, Mountain View, CA, USA). The DNA ladders were stained with ethidium bromide and DNA fragmentation was visualized under UV light.
Apoptotic cells were also determined by FACSCalibur Flow Cytometer (BD Biosciences, Sparks, MD, USA) using Annexin-V-FITC Apoptosis Detection kit (Calbiochem, San Diego, CA, USA).
In addition, HCC827 cells were stained with acridine orange dye mix and viewed using a fluorescence microscope. All experiments were repeated three times and a total of 200 cells were counted for each experiment.
Intracellular HCPT measurement. To measure intracellular HCPT accumulation, HCC827 cells treated with HTOFN or HCPT were harvested and lysed with DMSO. The cell extracts were sonicated and centrifuged, and the supernatant was then loaded onto HPLC for analysis.
Electrophoresis and immunoblotting. HCC827 cells lysed in RIPA buffer were subjected to SDS-PAGE/immunoblot analysis. The primary antibodies, occludin, claudin-1, claudin-2, claudin-3, claudin-4, claudin-7, ZO-1 and E-cadherin were obtained from Zymed (Invitrogen, Carlsbad, CA, USA), while all other antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). The proteins were detected by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ, USA).
Immunofluorescence light microscopy. After various treatments, HCC827 cells were fixed in cold methanol and were incubated with primary antibodies followed by incubation with secondary antibodies. The fluorescent signal was analyzed using a Zeiss AXIO IMAGER M1 microscope (Carl Zeiss MicroImaging Inc., Thornnwood, NY, USA).
Measurement of transepithelial electrical resistance. The HCC827 cells were plated on Transwell inserts and treated with HTOFN or TOFN. Transepithelial electrical resistance (TER) was determined by a Millicell-ERS voltohmmeter (Millipore Corp., Bedford, MA, USA).(11) All TER values were calculated by subtracting the resistance measured in the blank insert from the resistance measured in the insert with the monolayer and then multiplied by the surface area of the membrane. All experiments were repeated three times and the data were shown as the means ± SEM with an asterisk indicating P < 0.05.
HCPT drug-loaded Fe3O4 nanoparticles. We applied the unique water dispersible oleic acid-Triton X-100-coated Fe3O4 nanoparticles to load with HCPT. Transmission electron microscopy showed images of the successful drug-loaded nanoparticles (HTOFN) at low (a) and high (b) resolution, respectively (Fig. 1A). The average size of HTOFN is approximately 14 nm. Figure 1Ab shows the presence of a crystalline structure within the HTOFN core. X-ray diffraction (XRD) further demonstrated the XRD spectra of HTOFN (Fig. 1B), and no peak of impurity was observed, indicating that no new phases were formed and the crystalline structure of magnetite Fe3O4 nanoparticles did not change during the drug loading production. The peaks agreed with the standard Fe3O4 (cubic phase) XRD spectrum(12) and confirmed that the HTOFN cores were magnetite. Other methods such as Fourier transform infrared (FT-IR) spectroscopy and HPLC also provided quality assessment of the HCPT drug-loaded Fe3O4 nanoparticles (see Data S1 and Fig. S1 for details).
Increased cell apoptosis induced by HCPT drug-loaded Fe3O4 nanoparticles. To determine whether HTOFN can increase anti-cancer drug efficacy and promote cell death, we performed cytotoxicity assays using the human lung cancer cell line HCC827. The concentration of HCPT used to treat HCC827 cells was the same as that loaded in HTOFN. Under these experimental conditions, HTOFN significantly inhibited cell growth compared with HCPT treatment (Fig. 2A, HTOFN and HCPT). In addition, HTOFN demonstrated a sustained, dose-dependent anti-proliferative activity in HCC827 cells. Nanoparticles alone also had some effects in inhibiting cell growth compared with that of HCPT alone (Fig. 2A, TOFN and HCPT).
To determine whether cell growth inhibition was due to the apoptotic response, the DNA fragmentations were examined by agarose gel electrophoresis. When the HCC827 cells were treated with HTOFN, the intensity of fragmented chromosomal DNA bands was much higher than that observed from cells treated with TOFN or HCPT (Fig. 2B, lanes 1, 2 and 3, respectively). The formation of DNA ladders was clearly present after treatment with TOFN, but only weakly discernible when the cells were treated with HCPT. These results provide evidence that the remarkable enhancement of apoptosis was induced by the synergistic effect of Fe3O4 nanoparticles with HCPT in HTOFN on HCC827 cells.
Flow cytometry assays were carried out to verify the results obtained from DNA fragmentation experiments. Figure 2(C) shows that using the Annexin-V-FITC apoptosis detection method, HTOFN induced a much higher cell apoptosis rate than that of TOFN, HCPT or untreated control. We also observed an increase in the number of apoptotic nuclei in the HCC827 cells treated with HTOFN (Fig. 3A). Using acridine orange staining for apoptotic cells, apoptotic nuclei were identified by their distinctively marginated and fragmented appearance under the fluorescent microscope. We found that the percentage of apoptotic cells was 65.2%, 33.4% or 8.9% for HTOFN, TOFN or HCPT treatment, respectively (Fig. 3B). In summary, each of these experiments demonstrated a substantial increase in cell apoptosis following treatment of HCC827 cells with HTOFN.
Intracellular accumulation of HCPT. Drug-loaded nanoparticles (HTOFN) clearly inhibited cell growth and increased cell apoptosis. To understand the potential mechanism of this effect, HPLC was used to measure HCPT concentrations in HCC827 cells (Fig. 4). Our experimental results revealed that the concentration of HCPT was much higher in HTOFN-treated cells (Fig. 4D, peak 1) compared with that of TOFN or HCPT-treated cells (Fig. 4C,E, peak 1). Figure 4(F) showed that the accumulation of HCPT in HTOFN-treated cells was 58% of the HCPT (8.6 × 10−1 mg/L) loaded in HTOFN while the accumulation of HCPT in HCPT-treated cells was only 15% of 8.6 × 10−1 mg/L HCPT (the treatment dosage). These results indicate that HTOFN facilitated the uptake of the drug into HCC827 cells.
Activation of the caspase-8 pathway by HCPT drug-loaded Fe3O4 nanoparticles. To further explore the molecular mechanisms underlying the HTOFN-mediated apoptosis in HCC827 cells, we investigated apoptosis-related protein expression in HCC827 cells. When HCC827 cells were treated with HTOFN, the cleaved caspase-8 (p43/p41) signals on western blots were much stronger than those for cells treated with TOFN or HCPT alone (Fig. 5A). Similar results were obtained for cleaved caspase-9 and caspase-3. Poly (ADP-ribose) polymerase (PARP) is a known downstream target of active caspase-3 and can be cleaved during the induction of apoptosis. In HCC827 cells treated with HTOFN or TOFN, the cleavage of PARP via the proteolytic degradation of a full length PARP into the activated form was detected along with caspase-3 activation. Treatment of cells with HCPT at the concentration that was loaded in HTOFN did not initiate the activation of PARP above the control level (Fig. 5A). These data suggest that HTOFN treatment induces cell apoptosis by increasing activation of the caspase-8 pathway in HCC827 cells. Figure 5(B) summarizes the HTOFN-induced cell apoptosis activation signaling pathway.
Effects of HCPT drug-loaded Fe3O4 nanoparticles on cell–cell junction integrity. Functional cell–cell junction indicates the epithelial cell integrity. We first examined the subcellular distribution of TJ proteins by immunofluorescence light microscopy. In untreated epithelial cell monolayers, TJ integral membrane proteins, such as claudin-1, -3, -4, -7 and occludin, as well as TJ associate protein ZO-1, were all localized at the cell–cell junction (Fig. 6, control). The adherens junction marker, E-cadherin, was also concentrated at the cell–cell contact area. However, in HTOFN or TOFN-treated cell monolayers, all of the above proteins except occludin were internalized and the immunostaining signals were either reduced or disrupted (Fig. 6, HTOFN and TOFN).
We also examined TJ ultrastructure by TEM on HTOFN or TOFN-treated cells. In control HCC827 cells, the TJ was observed at the apical surface of adjacent cells as expected (Fig. 7Aa). However, TJ structure was often absent in the cells treated with HTOFN or TOFN (Fig. 7Ab,c).
To determine whether the TJ function had been affected, we measured the TER, a well-established method to assay the TJ barrier function of an epithelial monolayer. Our results revealed that TER was significantly decreased in the cell monolayers treated with HTOFN or TOFN, indicating that Fe3O4 nanoparticles reduced the TJ barrier function (Fig. 7B).
We finally sought to determine the expression of cell–cell junction proteins and the proteins of MAP kinase signaling pathways that are known to regulate cell–cell junctions in epithelial cells. The protein expression levels of claudin-1, -3, -4, -7, ZO-1 and E-cadherin were all decreased in a dose-dependent manner in the cells treated with HTOFN or TOFN (Fig. 8A). Claudin-2 was not expressed in HCC827 cells (data not shown). Interestingly, there was no significant change in the protein expression level of occludin after HTOFN or TOFN treatment, consistent with the subcellular localization result shown in Figure 6. Finally, we examined the signaling pathway changes after treatment with HTOFN or TOFN.
Among the MAP kinase family members, p38 MAP kinase was potently activated in a time-dependent fashion in HTOFN or HTOFN-treated cells (Fig. 8B). The stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) was also activated by HTOFN or TOFN treatment. In contrast, the phosphorylation of ERK1/ERK2 did not change following treatments, suggesting that the effects of treatments were potentially through the p38 or SAPK/JNK, but not through the activation of the ERK1/ERK2 MAP kinase signaling pathway.
In the present study, we have characterized a newly developed water-dispersible Fe3O4 nanoparticle-based formulation that can be loaded efficiently with the water-insoluble anti-cancer drug HCPT, a CPT analogue. The HCPT-loaded nanoparticles (HTOFN) demonstrated anti-proliferative activity in human lung cancer cells and also induced cell apoptosis through activation of the caspase-8 pathway.
Previously, the drug and nanoparticles were either conjugated chemically through the covalent bond or formed the bond through ionic interactions. However, the formation of the covalent bond might result in the difficulty of drug release while the binding through ionic interactions can result in drug dissociation from nanoparticles during the process of synthesis. In our nanomaterials, Fe3O4 nanoparticles were surrounded by the OA shell while Triton X-100 was anchored at the OA-water interface (Fig. S1). The HCPT was loaded between the OA shell and Triton X-100. This formulation could offer greater flexibility in terms of loading of the water-insoluble drug HCPT, as well as for efficient drug release. Our results indicated that OA is chemisorbed as a carboxylate headgroup on the surface of Fe3O4 nanoparticles. Thus, it is expected to provide better association of drug to nanoparticles with the surrounding OA shell acting as a drug reservoir. The OA shell can also protect Fe3O4 nanoparticles from oxidation and/or hydrolysis in the presence of water, which can significantly reduce the magnetization of the Fe3O4 nanoparticles.(4)
Triton X-100 is a nonionic detergent that is often used in biochemical applications to solubilize proteins.(13) We propose that the hydrophobic segments of Triton X-100 anchor at the interface of the OA shell around the Fe3O4 nanoparticles and the hydrophilic segments extend into the aqueous phase (Fig. S1). The OA shell surrounding the Fe3O4 nanoparticles plays a dual role serving as a drug reservoir as well as an interface for anchoring the hydrophobic chains of Triton X-100 to make the formulation water dispersible. Therefore, this formulation could facilitate the entry of an anti-cancer drug into cancer cells and enhance the accumulation of relevant molecules in the target cells.(14) Consistent with this hypothesis, results from our current study showed that the concentration of HCPT was significantly higher in HTOFN-treated cells than in HCPT-treated cells.
Apoptosis is an important biological process in many systems and can be triggered by a variety of stimuli.(15) The caspase family represents the key components of the apoptotic machinery within the cells and consists of at least 14 different caspase proteases in mammals.(16) Our results demonstrated that HTOFN treatments efficiently inhibited HCC827 cell growth and greatly increased cell apoptosis. When cells were treated with HTOFN they exhibited morphological features characteristic of apoptosis, such as membrane shrinkage and chromosomal condensation. Although some previous studies reported that nanoparticles could induce cell apoptosis, the underlying molecular mechanism was unclear.(17,18) We found that HTOFN treatments activated the caspase-8 pathway to induce apoptosis in HCC827 cells. Cleaved caspase-8 activated caspase-3 and caspase-9 that correlated with the increased cleaved PARP expression after HTOFN treatments. Although TOFN could also induce activation of the caspase-8 pathway compared with HCPT treatments and the control without treatment, the effects of TOFN alone on apoptosis were quite moderate when compared with that of HTOFN.
TJ is the most apical component of the junctional complex in epithelial and endothelial cells and separates the apical from the basolateral membrane and restricts the diffusion of ions and small molecules through the paracellular pathway.(19) It is well known that cell junctions are critical for maintaining normal epithelial cell functions and cell survival.(20) We found that HTOFN and TOFN disrupted TJ and the barrier function by downregulation of TJ as well as adherens junction protein expression. The localization of cell junction proteins was changed from the cell membrane to the cytoplasm after HTOFN and TOFN treatments. The reduced expression of TJ proteins was correlated with a significant increase in phosphorylation of p38 MAP kinase and SAPK/JNK in a time-dependent manner. The effect of TOFN on TJ disruption and p38 activation is most likely due to the oleic acid coated on TOFN because oleic acid has been reported to decrease TER and the barrier function, disrupt cell–cell contacts as well as activate the p38 MAPK.(21,22) Taken together, we propose that HTOFN and TOFN-induced TJ disruption is mediated through the activation of p38 and stress-related signaling pathways.
The authors thank Beverly G. Jeansonne and Joani T. Zary (Department of Anatomy and Cell Biology, East Carolina University Brody School of Medicine, North Carolina, USA) for their technical assistance. This research was supported in part by the National Institutes of Health Grants ES016888 and HL085752 (YHC), and CA111891 (QL), and from grants of the National Natural Science Foundation of China (90713023) and National Basic Research Program of China (2010CB732404) (XMW).
The authors have no financial interests in or financial conflict with the subject matter discussed in this manuscript.