In Situ-Induced Multivalent Anticancer Drug Clusters in Cancer Cells for Enhancing Drug Efficacy

Open AccessCCS ChemistryRESEARCH ARTICLE1 Apr 2019In Situ-Induced Multivalent Anticancer Drug Clusters in Cancer Cells for Enhancing Drug Efficacy Lingyun Zhou, Fengting Lv, Libing Liu and Shu Wang Lingyun Zhou Beijing National Laboratory for Molecular Sciences, Beijing (China) Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China) University of Chinese Academy of Sciences, Beijing 100049 (China) , Fengting Lv *Corresponding author: E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences, Beijing (China) Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China) , Libing Liu Beijing National Laboratory for Molecular Sciences, Beijing (China) Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China) and Shu Wang Beijing National Laboratory for Molecular Sciences, Beijing (China) Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China) University of Chinese Academy of Sciences, Beijing 100049 (China) https://doi.org/10.31635/ccschem.019.20180015 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Increasing intracellular drug concentration is an effective way for cancer chemotherapeutics to enhance efficacy and combat drug resistance. In this work, a series of anticancer drug conjugates were prepared by linking thiol-modified oligo(p-phenylene vinylene) with paclitaxel, vincristine, teniposide, tamoxifen, doxorubicin, or podophyllotoxin (OPV-S-Drugs) through a Michael addition reaction. These OPV-S-Drugs could undergo intracellular assembly and aggregation upon oxidation to yield multivalent anticancer drug clusters, which inhibited their diffusion from cancer cells. The intracellular aggregation of OPV-S-Drugs originates from π–π stacking and hydrophobic interactions between OPV backbones, followed by cross-linking via disulfide bond formation in the presence of reactive oxygen species (ROS). The drug clusters occur only in the cytoplasm of cancer cells expressing high ROS levels, but not in healthy mammalian cells, thus reducing the cytotoxicity to normal cells. Specifically, the super-toxicity of podophyllotoxin to normal cells was obviously suppressed while the drug efficacy was maintained through our new strategy. The diverse action mechanisms of OPV-S-Drugs toward cancer cells is proposed. Download figure Download PowerPoint Many efforts have been made to combat cancer, and fortunately, the rate of fatality from cancer is expected to decrease; however, the total number of people suffering from tumors and devitalized by cancer continues to increase.1–4 Multidrug resistance of cancer cells remains one of the most distressing issues all over the world.5–7 Patients who have seemingly recovered from chemotherapy can suffer from severe relapse due to the resistance of cancer cells to chemotherapy drugs. Many different chemical treatments have been studied and an antidrug resistance method among these is showing promise.8–14 Many new chemotherapeutics and techniques aimed at various tumor targets with increasing affinity and selectivity have been developed to combat the resistance of cancer cells, such as immunotherapy,15–19 photodynamic therapy,20–24 and ultrasonic therapy.25,26 Many drug delivery carriers have been prepared to specifically target and increase intracellular drug concentration to enhance drug efficacy against cancer cells.11,27,28 Recently, we have found that oligo(p-phenylene vinylene) with thiol groups (OPV-SH) is able to accumulate in cancer cells, but not in normal cells, because of the different levels of reactive oxygen species (ROS) inside tumor and normal cells. OPV-SH molecules respond to the intracellular ROS by forming disulfide bonds with each other and tightly clustering together. Once they form, bulky aggregations of OPV-SH cannot be easily expelled from cytosol through efflux proteins. The conjugation of paclitaxel (PTX) and OPV-SH (OPV-S-PTX) has the ability to selectively accumulate in cancerous cells, and it exhibits an enhanced ability to kill PTX-resistant cancer cells whilst maintaining a low toxicity to normal cells.29 In this work, we applied different types of chemotherapeutics to verify the expansive effect for OPV-S-Drugs. Vincristine binds with tubulin, the same target as PTX, not stabilizing but disassembling microtubules, which then induces the apoptosis of cancer cells.30,31 Tamoxifen, a medication for breast cancer, was also applied as it targets the estrogen receptor in cytosol.32–34 To verify whether the OPV-S-Drug exhibits a therapeutic function in the cell nucleus, teniposide and doxorubicin as topoisomerase inhibitors were also tested.35,36 From cytotoxicity tests for different kinds of OPV-S-Drugs, we found that most cancer cells expressing a high ROS level were more sensitive to the non-nucleus-targeting OPV-S-Drug, and for normal cells, the toxicity of the OPV-S-Drug was reduced in comparison with unmodified drug. It is well known that podophyllotoxin, a medication that destabilizes microtubules, has failed to act as antitumor drug due to the severe toxicity to normal cells.37,38 After conjugating podophyllotoxin with OPV-SH to form OPV-S-Podo, its cytotoxicity to the A549 tumor cell was enhanced, while exhibiting significantly reduced cytotoxicity toward normal cell lines. Our new strategy offers an alternative way to achieve small molecular drug delivery and assembly directly in the cytosol to form "un-expellable" bulky multivalent drug clusters. PTX, vincristine, teniposide, tamoxifen, doxorubicin, and podophyllotoxin were first introduced with a acryloyl or maleoyl group followed by a Michael addition reaction with OPV-SH to yield the conjugate, OPV-S-Drug. As illustrated in Scheme 1, except for tamoxifen and doxorubicin, acrylated drugs (Acr-PTX, Acr-Vin, Acr-Tenip, and Acr-Podo) were obtained by direct reaction of the individual drug with acryloyl chloride. To prepare acrylated tamoxifen (Acr-TMX), tamoxifen was first reacted with 2-bromoethanol and then with acryloyl chloride. Doxorubicin originally possesses an amino group, which could be linked with OPV-SH through succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Through a Michael addition reaction at room temperature, OPV-SH was covalently linked with Acr-Drug (Acr-PTX, Acr-Vin, Acr-Tenip, Acr-TMX, and Acr-Podo) and SMCC-Dox to yield OPV-S-Drugs (OPV-S-PTX, OPV-S-Vin, OPV-S-Tenip, OPV-S-Dox, OPV-S-TMX, and OPV-S-Podo). Scheme 1 | The synthesis routes for the modification of paclitaxel, vincristine, tamoxifen, podophyllotoxin, teniposide, and doxorubicin and the conjugation with OPV-SH. Download figure Download PowerPoint Intracellular assembly and aggregation of the OPV-S-Drug to form a bulky multivalent drug cluster was induced by intracellular ROS, which cancer cells are unable to expel (Figure 1a). Three factors were considered to drive the formation of the multivalent drug clusters specifically in cancer cells: (1) the self-assembly ability of the OPV-SH backbone by interchain π–π stacking and hydrophobic interactions; (2) the thiol groups in each OPV-SH preferentially formnig multiple disulfide bonds upon oxidation; and (3) the relatively higher ROS concentration in cancer cells. Thus, in cancer cells expressing high ROS level, the π–π stacking and hydrophobic-interaction-driven assembly gather the OPV-S-Drug molecules in cytosol and also bring multiple thiol groups in close proximity so that it is easy to induce numerous disulfide bonds to form bulky multivalent drug clusters. This mechanism was confirmed by dynamic light scattering (DLS) measurements. As shown in Figure 1b, each OPV-S-Drug is responsive to oxidative stress, in that they aggregated into much larger particles from nanometers up to micrometer in diameters. Figure 1 | (a) OPV-S-Drug assembly and aggregation in cancer cells. (b) Hydrodynamic diameter of OPV-S-Drugs with or without the addition of H2O2 measured by DLS (red: without H2O2 and blue: with H2O2). Download figure Download PowerPoint Intracellular ROS is one of the most important oxidants, since an excess of ROS can induce an imbalance in the cellular redox environment and cause oxidative stress. We measured the ROS expression levels in the A549, A549/T, MCF-7, MCF-7m, HCT-8, HCT-8/V, HPF, and MCF-10A cell lines to illustrate the different capabilities of these cells to oxidize the OPV-S-Drug. ROS capacity was defined as the ROS expression level presented by fluorescent intensity of dichloro-dihydro-fluorescein (DCFH) with the same amount for each cell line (). As shown in Figure 2a, the ROS capacities of the cancer cell lines (A549, A549/T, MCF-7, MCF-7m, and HCT-8/V) are higher than the normal cell lines (HPF and MCF-10A), which is in agreement with other reported works indicating that ROS expression of tumor cell lines is higher than normal cell lines.39 Although the samples of cancer cells studied did not show a uniformly high ROS capacity but had a broad range, HPF and MCF-10A cells exhibited a narrow distribution in the lower ROS capacity ranges. To obtain a more straightforward understanding of the ROS expression magnitude, confocal laser scanning microscope (CLSM) imaging was conducted on different types of cells incubated with dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Figure 2b). As intracellular ROS could oxidize nonfluorescent DCFH to fluorescent DCF, the fluorescence of DCFH was brighter as the amount of ROS produced increased. All cancer cells presented a brighter fluorescent signal compared with normal cell lines. A549/T and MCF-7m exhibit much higher brightnesses compared with other cells, indicating that drug-resistant cell lines express an even higher level of ROS. From both ROS capacity measurements and CLSM images, it is observed that HCT-8, a colon cancer cell line, shows a very low ROS expression compared with the normal cell lines, HPF and MCF-10A. The drug efficacy of OPV-S-Vin on HCT-8 also exhibited less significant improvement (). This result indicated that the enhancement of drug efficacy is highly dependent on the intracellular ROS concentration. Figure 2 | (a) ROS capacity of A549, A549/T, MCF-7m, HCT-8, HCT-8/V, HPF, and MCF-10A. (**p < 0.01 vs MCF-10A, ap < 0.01 vs HPF, bp < 0.05 vs HPF, one-way analysis of variance). (b) CLSM images of A549, A549/T, MCF-7m, HCT-8, HCT-8/V, HPF, and MCF-10A incubated with DCFH-DA (green) and Hoechst 33342 (blue). The scale bars are 20 μm. Download figure Download PowerPoint Except for doxorubicin, other drugs did not show obvious fluorescence under excitation with visible light; hence, OPV-S-Dox was used to investigate the localization and stability of the OPV-S-Drug conjugates. After incubation for 24 h, OPV-S-Dox mostly localized in cytosol (). The fluorescence from doxorubicin and OPV-SH co-localized quite well, which indicated that the OPV-S-Dox conjugate was stable in the intracellular environment. It was reported that the hydrolyzation of β-thiol ester happens quickly in an acid solvent or in the presence of an esterase, while the β-thiol ester bond is stable in a neutral pH solution.40 The cytotoxicity measurements of OPV-S-Drugs were carried out to confirm the enhancement of drug efficacy on cancer cells, the ability to combat drug resistance, and the reduction of cytotoxicity toward normal cells. As a control experiment, most of the pre-modified drugs (the Acr-Drugs and SMCC-Dox) presented sabotaged efficacy (). Dose–response curves of OPV-S-Drugs exhibited increased cell inhibition rates on most of the cancer cells. Moreover, for chemotherapeutic-resistant cell lines, OPV-S-Drugs also showed enhanced drug efficacy (). As shown in Table 1, the IC50 of PTX is 0.041 µM for regular A549 cells and more than 50 µM for drug-resistant A549/T cells. For OPV-S-PTX, the IC50 was reduced to 0.00033 µM for A549 cells and 6.3 µM for A549/T cells, which exhibited significant enhanced efficacy compared with that of PTX (Table 1; ). The cytotoxicity of OPV-S-PTX was reduced by a factor of 3 compared with PTX on the normal cell line HPF. Tamoxifen, as a selective estrogen-receptor modulator, is the preferred medication for treatment of breast cancer. As the breast cancer cell line MCF-7 and its multidrug-resistant cell line MCF-7m were reported to express high levels of estrogen receptor, these two cell lines were used to test the drug efficacy of OPV-S-TMX. The calculated IC50 of OPV-S-TMX on MCF-7 and MCF-7m is 16.7 and 19.0 μM, respectively, more than 1.5 times lower compared with tamoxifen citrate (29.3 μM for MCF-7 and 29.5 μM for MCF-7m; Table 1; ). MCF-10A was used as a normal breast cell line for comparison. OPV-S-TMX exhibited less toxicity compared with TMX on MCF-10A, which indicated that the OPV-S-TMX reduced the cytotoxicity to normal cell lines (Table 1; ). Vincristine works on the same target as PTX, but instead of stabilizing, it disassembles the microtubule. OPV-S-Vin showed less effect on HCT-8 cells, while exhibiting favorable enhanced drug efficacy toward the vincristine-resistant cell line HCT-8/V (IC50: 1.49 μM for vincristine and 0.27 μM for OPV-S-Vin; Table 1; ). This likely results from the relatively low ROS expression for HCT-8 cells causing less aggregation of OPV-S-Vin inside the cells. Table 1 | OPV-S-Drugs for drug-efficacy enhancement, toxicity reduction, and resistance combating toward different targeting cell lines. Without further explanation, A549, HPF, and MCF-7m cells were respectively used in the experiments of drug-efficacy enhancement, toxicity reduction, and resistance combating. IC50 was calculated through nonlinear curve fitting of dose-responsive line using the software of origin. As OPV-S-Dox and OPV-S-Tenip could not penetrate the nuclear envelope to react with DNA, it was essentially nontoxic for MCF-7 and MCF-7m cells. Podophyllotoxin, a microtubule destabilizer, was abandoned for use as an anticancer agent due to its poor selectivity, lower specificity, and high toxicity to normal cells and organs. Our intracellular aggregates strategy rearms the OPV-S-Drugs with less cytotoxicity to normal cells and combats multidrug resistance of cancer cells. We utilized this strategy on podophyllotoxin to improve its selectivity and reduce its toxicity to normal cells. As shown in Figure 3, OPV-S-Podo showed more efficacy to A549 cancer cells compared with podophyllotoxin (6.98 nM for OPV-S-Podo and 11.58 nM for podophyllotoxin); meanwhile, its cytotoxicity was obviously suppressed relative to the normal cell lines, HPF and MCF-10A. In the 8.0–38.0 nM concentration range outlined in the blue frame in Figure 3, the tumor cell A549 was effectively inhibited, while the growth of the normal cells HPF and MCF-10A was slightly influenced. This indicates that an intracellular aggregates strategy provides good potential for enabling the already banned, non-selective and less-efficient podophyllotoxin to potentially rejoin the arsenal of anticancer therapeutics. Figure 3 | Dose–response curves for cell viability of A549, MCF-10A, and HPF cell lines incubated with OPV-S-Podo for 48 h. The frame indicates the concentration range that OPV-S-Podo exhibits high toxicity to cancer cells but low toxicity to normal cells. Download figure Download PowerPoint The varying modes of action the OPV-S-Drugs has on cancer cells are summarized in Figure 4: OPV-S-Dox and OPV-S-Tenip: cannot penetrate the nucleus membrane and the drug efficacy is totally repressed (see case 1 in Figure 4); OPV-S-PTX, OPV-S-Vin, and OPV-S-Podo: multivalently bind to microtubules and induce cyto-apoptosis (see case 2 in Figure 4); and OPV-S-TMX: interacts with estrogen receptor and induces cyto-apoptosis (see case 3 in Figure 4). Figure 4 | OPV-S-Drug action modes in cancer cells. OPV-S-Dox and OPV-S-Tenip: cannot penetrate the nucleus membrane and the drug efficacy is totally suppressed (case 1); OPV-S-PTX, OPV-S-Vin, and OPV-S-Podo: multivalently bind to microtubules and induce cyto-apoptosis (case 2); and OPV-S-TMX: interacts with estrogen receptor and induce cyto-apoptosis (case 3). Download figure Download PowerPoint In summary, a series of drug conjugates were prepared by linking thiol-modified oligo(p-phenylene vinylene) and an anticancer drug through a Michael addition reaction. These OPV-S-Drugs could undergo intracellular assembly and aggregation upon oxidation to yield multivalent anticancer drug clusters, which inhibited their diffusion from cancer cells to improve efficacy and reverse drug resistance. The drug clusters occur only in the cytoplasm of cancer cells expressing a high level of ROS, but not in healthy mammalian cells, thus showing a reduction in the cytotoxicity to normal cells. This new strategy works well for non-nucleus-targeting anticancer chemotherapeutics, such as PTX, vincristine, teniposide, tamoxifen, doxorubicin, and podophyllotoxin. Moreover, on account of the ability of the OPV-S-Drugs system to reduce cytotoxicity to normal cells, podophyllotoxin was reconsidered as a potential antitumor agent. OPV-S-Drugs have been shown to provide new strategies to combat drug resistance, improve drug efficacy, and enrich the arsenal of anticancer chemotherapeutics, while simultaneously reducing the side effects on normal cells. Supporting Information Experimental procedures and additional are available online. Conflicts of Interest The authors declare no competing financial interests. 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