Gadolinium oxide nanoparticles enhance the cytotoxicity of chemotherapeutic drugs by blocking autophagic flux in cancer cells CURRENT

During chemotherapy, drugs can damage cancer cells’ DNA and cytomembrane structure, and then induce cell death. However, autophagy can increase the chemotherapy resistance of cancer cells, reducing the effect of chemotherapy. We found that gadolinium oxide nanoparticles (Gd2O3 NPs), which have great potential for use as a contrast agent in magnetic resonance imaging, could block the late stage of autophagic flux in a dose-dependent manner and then cause autophagosome accumulation in HeLa cells. When co-treated with 8 μg/mL Gd2O3 NPs and 5 μg/mL cisplatin, the number of dead cancer cells increased by about 20% compared with cisplatin alone. We observed the same phenomenon in cisplatin-resistant COC1/DDP cells. Thus, Gd2O3 NPs have significant potential for use in both cancer diagnosis and therapy.


Background
Cancer is a malignant invasion and uncontrolled growth of cells [1] and much energy and other resources are wasted at tumor sites. Chemotherapy is a common treatment for cancer; during chemotherapy, the chemical drugs can cause DNA damage and cell function failure [2], which can induce cell death [3].
In oncology, drug resistance is the dominant obstacle for therapy efficacy, and autophagy may help cancer cells to survive chemotherapy, to some extent, thereby leading to drug resistance [4]. In cells, material such as disabled organelles and proteins become coated by double-membranes in structures named autophagosomes [5]. Subsequently, the autophagosomes fuse with lysosomes, forming autolysosomes, in which the encapsulated contents are degraded into components for use in repair or cell structure formation. Cells perceive nanoscale materials to be foreign or aberrant, and these nanoscale materials can disturb the process of macroautophagy (defined in this article as autophagy) and cause lysosomal dysfunction [6]. Recently, our group reported that cadmium telluride quantum dots can block the late stage of autophagic flux in Saccharomyces cerevisiae and were toxic to the cells [7]. In addition, several other nanomaterials (such as fullerene C60 nanocrystals and MnO nanocrystals) have been reported to have potential to treat cancer by disturbing the autophagy process [8,9]. Thus, using nanoparticles (NPs) as chemosensitizers to induce disruptions to the 3 autophagic flux is a new method to treat cancer. The use of an autophagic inhibitor in combination with chemotherapeutic agents has already shown significant potential in cancer therapy [10,11].
Gadolinium oxide (Gd 2 O 3 ) nanoscale materials were developed to be used as contrast agents for magnetic resonance imaging (MRI) due to their longitudinal relaxivity characteristics [12]. They have significant potential for improving image quality. For example, terbium-doped Gd 2 O 3 NPs can be used as a fluorescence and MRI dual-modal contrast agent [13]. Due to the better T1 contrast enhancement compared to Gd3+ chelates, Gd 2 O 3 NPs are recognized as promising potential MRI T1 contrast agents [14,15]. The ability of Gd 2 O 3 NPs (which were used in this study) to enhance T1 contrast was previously reported [16].
Faucher et al reported that Gd 2 O 3 NPs could agglomerate in vesicles and be retained in glioblastoma multiforme cells [17]. Thus, the persistence of Gd 2 O 3 NPs may influence the cellular degradation system. Autophagy is a major intracellular degradation process that involves delivering cytoplasmic constituents sequestered in double-membrane vesicles to lysosomes [18], and it is particularly important for cells under certain environmental stress conditions [19]. Li

Preparation of Gd 2 O 3 NPs
The Gd 2 O 3 NPs were provided by Prof. Jinhao Gao [16]. The collected product was purified by dialysis. For the treatment experiments, cells were grown at an appropriate density in 96-or 6-well plates for 24 h to reach 80% population. Thereafter, the cells were cultured in fresh medium supplemented with the treatments (add rapamycin (Rapa, 20 μM), chloroquine (CQ, 100 nM), Gd 2 O 3 NPs (2, 4 or 8 μg/mL), cisplatin (5 or 10 μg/mL) alone or several of them together into the fresh medium).

Autophagy assay
Autophagosomes number changes were measured by monodasylcadaverine (MDC) staining, MDC is a selective fluorescent marker for autophagic vacuoles [21], to observe the autophagic process using fluorescence microscopy, Rapa (an agent that can induce autophagy) and CQ (a lysosomal inhibitor) were used as positive controls. And also measured by analysis of microtubule-associated protein light chain 3 (LC3) immunoblot, ratio of LC3-II/GADPH was used to quantification of LC3-II. Autophagic flux status was measured by analysis of p62 immunoblot.

Immunofluorescence
After treatment of the cells in 6-well plates for 24 h, the supernatants were removed. The cells were then fixed with 4% paraformaldehyde at room temperature for 20 min. Subsequently, 1 mL 0.5% Triton X-100 was added to each well, and the cells were left for 20 min. Thereafter, the cells were probed with primary rabbit polyclonal anti-LC3 antibody (Danvers, MA, USA, Cell Signaling Technology) overnight at 4 °C, and the secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China), was then added and the cells were incubated for 2 h at room temperature. Finally, after adding 1 mL 4′,6-diamidino-2-phenylindole (DAPI, final concentration 0.1 μg/mL) to each well and leaving the cells for 6 min at 37 °C, the cells were observed by confocal microscopy (Olympus, Tokyo, Japan). Samples were run on 15% SDS-polyacrylamide gel electrophoresis (PAGE) gels and transferred onto polyvinylidene fluoride (PVDF) membranes. Each membrane was blocked with 5% skimmed milk and 0.1% Tween-20 overnight at 4°C and probed with LC3 or p62 or GAPDH primary antibody for 2 h.

Protein extraction and immunoblot analysis
Regarding the secondary antibody, the incubation was performed with horseradish peroxidaseconjugated IgG for 2 h. Blots were developed using a chemiluminescent horseradish peroxidase substrate (Merck Millipore, Billerica, MA, USA). Each Western blot was analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).

Cell viability assay
Cell viability was measured using a (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After treatment, medium supplemented with MTT solution was added to each well and the cultures were incubated for 4 h at 37 °C. Thereafter, the supernatants were removed and 100 μL dimethyl sulfoxide (DMSO) was added to the plates, which were then incubated for 10 min at 37 °C.
The absorbance at 595 nm was detected using a microplate reader (Thermo, Grand Island, NY, USA).

Statistical analysis
Each experiment was performed in three replicates, and the values shown represent the means ± standard deviation. Significant differences between the treatments were determined by Student's ttest using SigmaPlot Version 12.5 (Systat Software, San Jose, CA, USA).

Gd 2 O 3 NPs cause autophagosome accumulation in HeLa cells
To investigate the effect of Gd 2 O 3 NPs on autophagy, firstly, we used MDC staining, after treatment of HeLa cells with 4 and 8 μg/mL Gd 2 O 3 NPs, we observed increased numbers of MDC-labeled vesicles compared to in the control cells, and this effect was dose-dependent (Fig. 1A). This indicated that Gd 2 O 3 NPs could induce the accumulation of autophagic vacuoles in HeLa cells.
In detail, immunofluorescence and immunoblotting analysis of LC3 were conducted. LC3 is the most widely used biomarker for autophagosomes [22], and the amount of LC3-II has a positive correlation with the autophagy level [23]. As shown in Fig. 1B, 4  Autophagosome accumulation is caused by inhibition of late-stage autophagic flux rather than induction of autophagy Accumulation of autophagosomes could be the result of induction of autophagy or inhibition of autophagosome clearance [24]. To determine what the case for Gd 2 O 3 NPs was, we measured the turnover of LC3 in the presence or absence of 100 nM CQ and 20 μM Rapa, respectively. Rapa caused LC3-II accumulation as expected and adding CQ further increased the Rapa-induced LC3-II accumulation. No increase in LC3-II accumulation was found in cells co-treated with 8 μg/mL Gd 2 O 3 NPs and CQ compared to cells treated with Gd 2 O 3 NPs alone ( Fig. 2A and B). This indicated that Gd 2 O 3 NPs caused autophagosome accumulation by inhibiting autophagosome clearance rather than by inducing autophagy.
To further confirm this conclusion, autophagic flux was monitored by measuring p62, a specific substrate that is preferentially degraded by autophagy [25]. As expected, as shown in Fig. 2C  as assessed by MTT assay (Fig. 3B). The microscopy images support this conclusion, with more cells dying when co-treated with chemical drug and 8 μg/mL Gd 2 O 3 NPs compared with each agent alone (Fig. 3C).
Autophagic blocking contributed to enhanced cytotoxicity 8 Autophagy is a major method of cancer cells to combat chemical drugs. Thus, to find out whether the blocked autophagic flux caused by Gd 2 O 3 NPs is responsible for the enhanced cytotoxicity, we assessed how low-dose Gd 2 O 3 NPs affect the cell death caused by 5 μg/mL cisplatin, a frequently used chemotherapeutic agent. Cell death induced by cisplatin is known as apoptosis [26]. For quantification of apoptotic cells, the HeLa cells were stained with Annexin V-FITC and PI after 24 h of treatment. The results showed that the combination of 8 μg/mL Gd 2 O 3 NPs and 5 μg/mL cisplatin caused twice the apoptotic cell number compared to 5 μg/mL cisplatin alone. Treatment with 8 μg/mL Gd 2 O 3 NPs alone did not cause a significant increase in the number of apoptotic cells (Fig. 4A).
To find out whether the blockage of the autophagic flux is responsible for the cell death, we replaced Gd 2 O 3 NPs with 100 nM CQ in the co-treatment of HeLa cells with cisplatin. We then estimated the number of apoptotic cells as we had done before. The number of apoptotic cells caused by the combination of 100 nM CQ and 5 μg/mL cisplatin was increased by about 100% than the number caused by 5 μg/mL cisplatin alone (Fig. 4B). These results indicated that Gd 2 O 3 NPs can enhance cisplatin-mediated cell death.
To provide more proof, 20 μM Rapa was used to recover the autophagic flux impaired by Gd 2 O 3 NPs.
As shown in Fig. 5, not only did 20 μM Rapa decrease the level of p62 accumulation induced by 8 μg/mL Gd 2 O 3 NPs (Fig. 5C and D), indicating that Rapa can indeed recover autophagic flux impaired by Gd 2 O 3 NPs, but Rapa also significantly reduced the chemosensitization caused by Gd 2 O 3 NPs. With the addition of Rapa, about 20% more cells survived when co-treated with Gd 2 O 3 NPs and cisplatin ( Fig. 5A and B). These results indicated that inhibition of autophagic flux is required for chemosensitization induced by Gd 2 O 3 NPs.

Gd 2 O 3 NPs also enhance chemosensitization in cisplatin-resistant cancer cells
Some tumor cells develop resistance to chemotherapeutics via autophagy; thus, inhibiting autophagy could be a promising strategy for cancer therapy [11]. To assess whether the sensitization effect is HeLa-specific, we evaluated the chemosensitization effect of Gd 2 O 3 NPs in cisplatin-resistant cancer cells, COC1/DDP, using an Annexin V/PI assay. Similar to the situation with HeLa cells, the combination of 8 μg/mL Gd 2 O 3 NPs and 10 μg/mL cisplatin resulted in a lower cell viability ( Figure S1), compared to treat with cisplatin alone, number of dead cells increased by about 100% (Figure 6). This result indicated that the chemosensitization effect of Gd 2 O 3 NPs was also efficient in COC1/DDP cells.

Discussion
The most difficult factor that we confront when treating cancer is drug resistance among cancer cells [27], and autophagy is an important reason for drug resistance. Several nano-materials have been found to induce cancer cell death by causing autophagy failure [28]. Moreover, due to the enhanced permeability and retention (EPR) effect at tumor sites, NPs mainly become distributed at tumor sites [29], which means that NPs could improve the efficacy of chemotherapy. Nanomaterials such as C60 and MnO nanocrystal may kill cancer cells by inducing autophagy, and they have the potential for use in cancer therapy [8,9].
In this study, we found that Gd 2 O 3 NPs had a strong ability to block autophagic flux and could result in cancer cell death when used with chemical drugs to treat cancer cells. We were convinced that the autophagic response induced by Gd 2 O 3 NPs increased with the concentration of Gd 2 O 3 NPs (Fig. 1), and the chemosensitization effect of Gd 2 O 3 NPs also increased (Fig. 3B) Not applicable. This manuscript does not report on or involve the use of any animal or human data or tissue.

Consent for publication
Not applicable. This manuscript does not contain data from any individual person.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.  nM CQ caused the amount of p62 to be decreased and increased, respectively, and Gd2O3 NPs increased the amount of p62 in a dose-dependent manner. (D) Quantification of (C).

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