Emodin-loaded polymer-lipid hybrid nanoparticles enhance the sensitivity of breast cancer to Doxorubicin by inhibiting epithelial mesenchymal transition

The role of epithelial mesenchymal transition (EMT) involved in breast cancer metastasis and chemoresistance has been increasingly recognized. However, it’s necessary to search for more effective strategies to inhibit EMT thereby increase the sensitivity of breast cancer cells to chemotherapy drugs. Emodin has a potential in overcoming tumor drug resistance and restraining the development of EMT, but the poor internalization into breast cancer cells limited the application. MCF-7/ADR cells have more EMT characteristics than MCF-7 cell. EMT in MCF-7/ADR cells promotes the development of drug resistance via apoptosis resistance and facilitating the expression of P-gp. The anticancer effect of DOX enhanced by the decreasing of drug resistance protein P-gp and apoptosis related proteins after EMT inhibited in MCF-7/ADR cells. E-PLNs increase the cellular uptake of EMO and restore DOX sensitivity in MCF-7/ADR cells by inhibiting EMT.


Abstract Background
The role of epithelial mesenchymal transition (EMT) involved in breast cancer metastasis and chemoresistance has been increasingly recognized. However, it's necessary to search for more effective strategies to inhibit EMT thereby increase the sensitivity of breast cancer cells to chemotherapy drugs. Emodin has a potential in overcoming tumor drug resistance and restraining the development of EMT, but the poor internalization into breast cancer cells limited the application.
Results MCF-7/ADR cells have more EMT characteristics than MCF-7 cell. EMT in MCF-7/ADR cells promotes the development of drug resistance via apoptosis resistance and facilitating the expression of P-gp. The anticancer effect of DOX enhanced by the decreasing of drug resistance protein P-gp and apoptosis related proteins after EMT inhibited in MCF-7/ADR cells. E-PLNs increase the cellular uptake of EMO and restore DOX sensitivity in MCF-7/ADR cells by inhibiting EMT. Conclusion E-PLNs inhibit EMT to enhance the sensitivity of breast cancer to DOX. The combination of E-PLNs and DOX can improve the e cacy of DOX in the treatment of breast cancer, which provides a new method to prevent or delay clinical drug resistance.

Background
Despite signi cant advances in cancer diagnosis and treatment, breast cancer to be the number one cancer in the world by 2020, more than 90% of the death causes of breast cancer patients are related to chemotherapy resistance , Sung et al. 2021. The role of epithelial mesenchymal transition (EMT) involved in chemotherapy resistance has been increasingly recognized. Asiedu MK and his colleagues found that EMT induced by TGF-β pathway in breast cancer cells decreased the sensitivity to Doxorubicin (DOX) and paclitaxel (Asiedu et al. 2014). Reversal tumor drug resistance through EMT inhibition is a new strategy and search for a safe and effective EMT inhibitor is necessary.
Emodin (EMO), the active component of Rheum rhabarbarum, is proved to have antitumor activity in breast cancer (Sun et al. 2015). In addition, EMO has a potential in overcoming tumor drug resistance and restraining the development of EMT (Liu et al. 2020, Fu et al. 2012, Lu et al. 2016, Gu et al. 2019. It can effectively restore the effect of DOX, vincristine and other anticancer drugs by downregulating the expression of P-gp in MCF-7/ADR cells. It was found that the combination of DOX and EMO effectively inhibit the expression of snail, twist and slug, thereby inhibiting cell proliferation and promote cell apoptosis. This combination results the suppression of cell migration and invasion. However, the poor internalization into the breast cancer cells limited the application of EMO. Hence, improving EMO internalization in breast cancer cells tend to be a reliable way to enhance the anti-cancer effect of DOX. Furthermore, the drug reversal effect of EMO whether rely on the repression of EMT still needed to be elucidated. Nanoparticles loaded with anticancer drugs become an ideal formulation for reversing chemoresistance (Li et al. 2016, Markman et al. 2013. The tumor targeting effect of nanoparticles is basely depended on the enhanced permeability and retention (EPR) in tumor microenvironment (TME), thus drug accumulation increased in TME (Kalyane et al. 2019). Polymeric lipid nanoparticles (PLNs), designed to combine the advantages of both liposomes and polymeric nanoparticles, can meet the structural and property differences of various anticancer drugs, as well as the combination therapy strategy, making them a promising drug carrier (Jain et al. 2020, Mukerjee, Ranjan andVishwanatha 2012).
Targeting EMT is becoming one of the new strategies for drug resistance reversal in various tumors. In this study, the relationship of EMT and drug resistance were discussed. Here, we provided evidence for E-PLNs to improve the therapeutic effect of DOX on breast cancer. Further, the evidence concerning drug resistance reversal mechanism of E-PLNs in suppressing EMT is provided.

Results
EMT is associated with DOX resistance in breast cancer cells We compared the effect of DOX on the viability of MCF-7 cells and MCF-7/ADR cells. As Fig. 1A, the toxicity of DOX on MCF-7/ADR cells than MCF-7 cells were less, IC50 was 5.35 μM, while the IC50 of MCF-7/ADR cells were 124.1 μM. MCF-7/ADR cells were resistant to DOX with a resistance index of 23.2.
EMT is characterized by loss of E-cadherin expression and upregulated Vimentin and N-cadherin ( Fig. 1B and C). DOX induced drug resistant cells have EMT characteristics, and the ability of invasion signi cantly enhanced ( Fig. 1D and E).

EMT inhibiting cell proliferation and invasion
TGF-beta signaling has been shown to play an important role in EMT. Galunisertib, a selective TGF-β receptor type I (TGF-βRI) kinase inhibitor to block TGF-β signaling reverse EMT, is applicated in this part to investigate the relationship of EMT, cell proliferation and invasion. It can be seen from Fig 2A that different concentrations of Galunisertib can inhibit the proliferation of MCF-7/ADR cells, and the IC50 value calculated is 248.2 μM. When the concentration of Galunisertib was 30 μM, the cell viability was more than 90%. Therefore, the concentration of 30 μM Galunisertib is selected to application for the subsequent experiment to exclude the interference of Galunisertib cytotoxicity. Galunisertib reversed EMT marker protein expression in the selected concentration ( Fig. 2B and C). The therapeutic effect of DOX was improved under the incubation of 10 μM DOX and 30 μM Galunisertib (Fig. 2D) MCF-7/ADR cells have higher invasive ability than the DOX sensitive MCF-7 cells, but invasive ability of MCF-7/ADR cells treated with DOX did not change. Compared with DOX (10 μM) group, Galunisertib (30 μM) group cells was decreased and the invasion activity was inhibited. When Galunisertib and DOX were used together, the inhibition effect was stronger ( Fig. 2E and F). These results suggest that the inhibition of EMT may weaken the drug resistance of MCF-7/ADR cells.

Blocking EMT restrain the expression of P-gp and induced apoptosis
Apoptosis is an important manifestation of anti-tumor effect of chemotherapy drugs. We can see from Fig 3A and B that the apoptosis rate of DOX (10 μM) group is only 13.81%, Galunisertib (30 μM) group (the apoptosis rate is 7.85%) has little effect on apoptosis. When DOX was combined with Galunisertib, the number of apoptotic cells increased, and the apoptotic rate was 23.88%. It can be seen from Fig. 3C and D that compared with DOX group, Bax/Bcl-2 ratio increased in DOX+Galunisertib (30 μM) group. These results suggest that inhibition of EMT can enhance DOX induced apoptosis in MCF-7/ADR cells.
EMT was closely related to the process of drug resistance, one of which was upregulation of drug resistance related transporters. As can be seen in Fig. 3E and F, the difference of expression level of P-gp protein in Control group and DOX group was not signi cant. However, when MCF-7/ADR cells were treated with Galunisertib, the expression levels of P-gp protein were down-regulated. More importantly, the expression of P-gp in DOX+Galunisertib group was signi cantly suppressed than DOX group, resulting in decreasing of DOX e ux. Therefore, blocking EMT down-regulate the expression level of P-gp, thereby restraining the DOX e ux in MCF-7/ADR cells.
E-PLNs increase the cellular uptake and cytotoxity of EMO E-PLNs prepared by nano precipitation method are light yellow liquid (Fig. 4A). In order to preserve the samples for a long time, we keep E-PLNs samples through freeze-drying (Christ ALPHA 1-4 LD plus). The particle size of E-PLNs was 122.7±1.03 nm (Fig. 4C), zeta potential was -28.5±1.55 mV (Fig. 4D) and PDI was 0.118. The results show that the nanoparticles have uniform size and good stability.
As E-PLNs have no uorescence, the C6-PLNs, characterized by green uorescence, is formulated to analyze the cellular uptake of PLNs carriers. After the coumarin-6-labeled nanoparticles were internalized, the green uorescence mainly distributed in the cytoplasm (Fig. 5A), indicating that PLNs carriers promote the cellular uptake of the loaded drugs. Furthermore, ow cytometry quantitatively analyzes cellular uptake, and the uptake intensity was expressed as the average uorescence intensity of cells. The average uorescence intensity of cells was 500, indicating that the cells had a high uptake of nanoparticles ( Fig 5B and C). The cytotoxicity of EMO group (Fig.5D) and E-PLNs ( Fig.5E) group on MCF-7/ADR cells gradually increased with the increase of EMO concentration. The IC50 of EMO and E-PLNs were 150.5 μM and 138.7 μM, respectively, which indicated that E-PLNs could enhance the antitumor activity of EMO. In order to further explore whether EMO or E-PLNs can enhance the toxicity of DOX, a suitable concentration was selected and combined with DOX to detect the effect on cell viability (Fig.5F). We selected 30 μM EMO and E-PLNs for the subsequent combined administration experiment. It can be seen from Fig. that 30 μM E-PLNs can enhance the cytotoxicity of different concentrations of DOX. Finally, 10 μM DOX was selected to explore how E-PLNs improve the therapeutic effect of DOX.

E-PLNs enhance the effect of DOX in apoptosis induction
MCF-7/ADR cells were treated with DOX, DOX+EMO, DOX+E-PLNs to investigate whether E-PLNs induced cell apoptosis. The morphological changes of apoptotic cells are shown in Fig 6A. We observed that the untreated cells were round or oval blue nuclei, while the cells in the treatment group were fragmented and apoptotic cells were formed, and the number of apoptotic bodies was signi cantly reduced in the E-PLNs group. It can be seen from Fig. 6B and C that compared with DOX (10 μM) group (15.52%), the apoptosis rates of DOX+EMO (30 μM) and DOX+E-PLNs (30 μM) groups were 18.49% and 24.48%, respectively, and the apoptosis rate was signi cantly increased. Western blot results also showed that the ratio of Bax/Bcl-2 was increased and the expression of Fas was up-regulated in DOX+EMO (30 μM) and DOX+E-PLNs (30 μM) groups, which promoted cell apoptosis. At the same time, the expression level of Bcl-2 was down regulated. DOX+E-PLNs group showed a more signi cant effect on the expression of apoptotic protein ( Fig. 6D and E). Flow cytometry combined with Western blot showed that E-PLNs may enhance the apoptosis inducing effect of DOX on MCF-7/ADR cells through affecting apoptosis related pathways.

E-PLNs enhance DOX accumulation and restrain the expression of P-gp
The accumulation of DOX in MCF-7/ADR cells treated by different administration groups (Control, DOX, DOX+EMO, DOX+E-PLNs) was analyzed quantitatively by ow cytometry (Fig. 7A). From Fig. 7B, it can be seen that the peak map of DOX+EMO (30 μM) and DOX+E-PLNs (30 μM) groups shifts to the direction of high uorescence intensity compared with DOX (10 μM) group. The average uorescence intensity of DOX+E-PLNs is higher than that of DOX group indicated that E-PLNs promote the uptake of DOX and increase the DOX accumulation in MCF-7/ADR cells. In addition, the expression of P-gp signi cantly decreased in DOX+EMO and DOX+E-PLNs groups compared with control and DOX groups (Fig 7C and D), indicated that E-PLNs could effectively inhibit the expression of P-gp protein thus enhancing the effect of DOX.

E-PLNs inhibit the expression of EMT marker protein and cell invasion
The expression of E-cadherin decreased, while the expression of N-cadherin and Vimentin increased in MCF-7/ADR cells, which indicated the occurrence of EMT. It can be seen from Fig. 8A and B that there is no signi cant difference in the protein levels of E-cadherin and Vimentin between the Control group and the DOX group, but the protein level of N-cadherin increases signi cantly, which indicates that DOX administration to MCF-7/ADR cells may further promote EMT. In DOX+E-PLNs (30 μM) group, the expression of E-cadherin protein was increased, and the expression of vimentin and N-cadherin protein was up-regulated. These results indicate that E-PLNs can effectively inhibit EMT, and the enhancement of DOX sensitivity of MCF-7/ADR cells by E-PLNs is related to the inhibition of EMT.
Cell invasion is an important manifestation after EMT induction. In Fig. 8C and D, there was no signi cant difference in the number of cell invasion between the Control group and DOX group. Compared with DOX group, the number of invasive cells in DOX+EMO (30 μM) group and DOX+E-PLNs (30 μM) group decreased, and DOX+E-PLNs group was more signi cant. These results shown that the combination of E-PLNs and DOX can inhibit the occurrence of EMT and the tendency to invasion in MCF-7/ADR cells.

Discussion
Chemotherapy is the main treatment for early stage of breast cancer, but the sensitivity of tumor cells to chemotherapeutic drugs decreases with long-term chemotherapy. Increasing evidence shown that EMT is involved in the development of drug resistance in breast cancer (Jiang et al. 2017). After EMT was induced by snail eukaryotic expression vector, the expression of BCRP were signi cantly increased and the drug resistance of MCF-7 cells was increased to 9.93 times (Du and Shim 2016). In this article, we nd that appears MCF-7/ADR cells have more signi cant characteristics of EMT than DOX sensitive MCF-7 cells, manifested as the down-regulated of E-cadherin protein and the up-regulated of N-cadherin protein and Vimentin. In fact, Li and his colleagues also con rmed that DOX could induce EMT and apoptosis in MCF-7 cells (Li et al. 2020b). The invasion and metastasis are signi cantly enhanced in tumor cells with EMT characteristics. Indeed, the invasion test showed that MCF-7/ADR cells had more invasive cells than MCF-7 cells, which proved that MCF-7/ADR cells had stronger invasion ability.
Transforming growth factor-beta (TGF-β) signaling plays a key role in epithelial-mesenchymal transition (EMT) of tumors (Xu, Lamouille and Derynck 2009). Galunisertib is a selective TGF-β receptor type I (TGF-βRI) kinase inhibitor which blocking TGF-β signaling reverse EMT (Xu et al. , Rodón et al. 2015). The direct toxicity of DOX to cells was enhanced by Galunisertib, which decreased cell viability and increase cell invasion. Moreover, the apoptosis induction in MCF-7/ADR cells of DOX restored after EMT suppression. In addition, previous studies have shown that both P-gp and ATP binding cascade transporters are regulated by EMT. In this study, as the number of cells with EMT characteristics were decreased, and the expression of P-gp protein was restrained. Therefore, it is worth to con rm that EMT participates in the drug resistance of cancer cells by regulating the processes of apoptosis and drug resistance proteins expression. EMO has been found to inhibit to increase sensitivity to DOX and PTX in breast cancer (Li et al. 2020b, Gu et al. 2020, and inhibits EMT in triple negative breast cancer (Song et al. 2018). It's reported that solid lipid nanoparticles of EMO can signi cantly block the cell cycle and induce apoptosis of MCF-7 cells compared with free EMO (Wang et al. 2012), which indicates that lipid nanoparticles delivering EMO may be a promising method for cancer treatment. In this research, we prepared PLNs encapsulated EMO to enhance cellular uptake. As E-PLNs have no uorescence, the C6-PLNs is formulated to analyze the cellular uptake of PLNs carriers. The result shown that C6-PLNs can be absorbed by MCF-7/ADR cells, and quantitative analysis of the average uorescence intensity shown that PLNs structure improve the uptake of C6. Furthermore, the cellular uptake and anti-tumor effect of DOX improved with the combination of E-PLNs, indicated that EMO enhanced the therapeutic effect of DOX on tumor cells. The main mechanisms of the anti-cancer effect in DOX is apoptosis induction, and the apoptosis rate increased to 24.48% combined with E-PLNs. The apoptotic pathway was activated by the increase of Bax/Bcl-2 ratio. The expression of P-gp is restrained, resulting in the decrease of DOX e ux. The upregulated expression of E-cadherin, and down-regulated N-cadherin and Vimentin in MCF-7/ADR cells incubated with E-PLNs indicate that E-PLNs inhibit EMT, therefore reverse DOX resistance in MCF-7/ADR cells.
In this study, we nd that EMT is involved in drug resistance, which is related to the regulation of apoptosis and the expression of drug resistance transporters. EMT is one of the key factors to induce DOX resistance in breast cancer. We also found that the expression of E-cadherin in DOX+E-PLNs group was higher than that in DOX group, while the expression of Vimentin and N-cadherin was down regulated. In addition, when E-PLNs were combined with DOX, the number of invasive cells decreased signi cantly. These results suggested that E-PLNs enhancing the sensitivity of breast cancer to DOX depended on inhibiting EMT.

Conclusion
Drug resistance in tumor is a phenomenon which the continuous administration of chemotherapeutic drugs induces drug tolerance. EMT is one of the possible ways causing DOX resistance in breast cancer. Our data suggested that the DOX resistance in breast cancer is associated with EMT. E-PLNs enhance the sensitivity of DOX in breast cancer via the suppression of EMT. We con rmed that E-PLNs enhance the effect of DOX, which is closely related to the regulation of EMT thereby inhibiting of apoptosis resistance and P-gp expression. The combination of E-PLNs and DOX provides a new scheme for clinical treatment. It's potential that E-PLNs turn into an adjuvant therapy drugs of breast cancer in the feature. Further investigations in animal models and mechanism are warranted.

Preparation of E-PLNs lyophilized powder
We prepared E-PLNs by nano precipitation method (Tahir et al. 2019). Brie y, EMO and PLGA were dissolved in acetone. Another soybean phospholipid and DEPE-PEG 2000 were dissolved in 5% (W/V) F68 aqueous solution. The organic phase was slowly and evenly added to Pluronic F-68 aqueous solution at 75 ℃, and stirred (8000 rpm for 40 min) until the acetone completely evaporated. The supernatant was collected by centrifugation (1000 rpm for 5 min). The particle size and Zeta potential of E-PLNs were measured by Malvern Zetasizer Nano ZS90. The prepared E-PLNs were added with 10% (W/V) freezedrying protective agent (Mannitol). After being frozen overnight at -80 ℃, E-PLNs were freeze-dried for 24 h and stored at 4 ℃.
Cell culture MCF-7 cells were cultured in 10% FBS of RPMI-1640 medium under de ned circumstances. In order to maintain cell resistance to DOX, MCF-7/ADR cells were cultured in RPMI-1640 medium containing DOX (250 ng/mL) every third passage.

Cellular uptake
Coumarin-6 (200 µg/mL) was encapsulated in PLNs instead of EMO to prepare coumarin 6-PLNs (C-6 PLNs). MCF/ADR were incubated with C-6 PLNs for 2 h, xed by 4% paraformaldehyde, and treated with Hochest staining solution for 5 min. The uptake of C-6 PLNs in MCF/ADR was observed under laser confocal microscope and analyzed by Flow cytometry.

MTT assay
Cells were cultured in 96 well plate for 24 h. Then the fresh culture medium containing free DOX, free EMO, E-PLNs, DOX+EMO, DOX+E-PLNs were added to the culture medium, and cultured for more than 48h for cell test. After incubation with MTT (5 mg/mL, 20 μ L/well) for 4 h, the supernatant was removed. The cells were exposed to DMSO solution (150 μL/well) and incubated for 15 min. Finally, the OD value was detected (Microplate Reader, 490 nm) and cell viability was calculated.

Cell invasion assay
Cells were resuspended in RPMI-1640 medium without FBS, then seed in the upper chamber for 24 h, and 500 μL culture medium containing different drugs was added into the lower chamber. Cells were xed with 4% paraformaldehyde for 10 min and stained with 200 μL crystal violet staining solution for 15 min. 5 elds of vision were randomly selected under a 100 × microscope to observed the cells passing through the lter membrane. After taking photos, 500 μL 33% acetic acid solution was added, shaking for 10 min, the OD value was measured to indirectly re ect the number of invasive cells.
Apoptosis study MCF-7/ADR cells (3×105 cells/well, 6 well plate) were cultured, then treated with fresh medium containing different drugs for 48 h. The cells were digested with trypsin without EDTA. After centrifugation again, cells were suspend with 500 μL Binding Buffer. Then, Annexin-V/FITC and PI staining solution was added with 5 μL each and mixed thoroughly. All experimental groups were sincubated in the dark for 15 min. Finally, the apoptosis was detected on the ow cytometer.

Western blotting analysis
Treated with different treatments for 48 h, cells were lysed with lysate, and the protein was obtained by centrifugation (10000 rpm for 5 min). BCA method was used to determine the protein concentration. After the expressed proteins (30 μg each) were separated by SDS-PAGE (Bio RAD), and transferred to PVDF membrane. The unbound protein sites were blocked with 5% skim milk for 2 h. The PVDF membrane was incubated with solution containing with different primary antibodies at 4 ℃ overnight. After washed with PBST for 1 h, The PVDF membrane incubated with solution containing horseradish peroxidase bound secondary antibody for 2 h. The PVDF membrane was evenly coated with ECL developer. According to the manual, the exposure was carried out with chemiluminescence imaging system and analyzed with Image J software.

Statistical analysis
The experiment was repeated three times, and the average value ± SD value was calculated. GraphPad Prism software analyzes and draws pictures, and the ONE-WAY ANOVE analysis is used for signi cance. * p <0.05, ** p <0.01, and *** p <0.001 are considered to be signi cantly different.

Declarations
Ethics approval and consent to participate Not Applicable.

Consent for publication
All authors agree with the publication of this manuscript in this journal.

Availability of data and materials
The data will be available if needed.

Competing interests
The authors declare that there is no con ict of interest Funding This work was supported by the Natural Science Foundation of Guangdong (2021A1515011241).

Figure 3
Effect of Galunisertib combined with DOX on apoptosis and expression of drug resistance protein. (A) and (B) The apoptotic rate of MCF-7/ADR cells treated with different groups (DOX, Galunisertib, DOX+ Galunisertib) was measured by ow cytometry. (C) and (D) The expression of Bax and Bcl-2 in MCF-7/ADR cells treated with DOX, Galunisertib, DOX+Galunisertib. Relative levels of the proteins were quanti ed by densitometric analysis. (E) and (F) The expression of P-gp and MRP1 in MCF-7/ADR cells treated with different groups (DOX, Galunisertib, DOX+Galunisertib) . N=3, **p < 0.01, ***p < 0.001 compared with DOX group.

Figure 4
Page 17/21 The characterization of E-PLNs. (A) The morphology of E-PLNs. (B) Lyophilized powder of E-PLNs. (C) and (D) The particle size and Zeta potential of E-PLNs was measured by the laser particle size analyzer.