Association of MTDH expression with the probability of disease-free survival and overall survival and efficacy of TAX treatment in patients with breast cancer
In a cohort of 44 neoadjuvant chemotherapy breast cancer patients, 29 patients were MTDH gene positive and 15 patients were negative. Patients with high MTDH protein expression (tan or brown staining in the cell membrane or cytoplasm, Fig. 1a) had significantly worse probability of disease-free survival (DFS) and overall survival (OS) than those with low MTDH protein expression (Fig. 1c, d). Furthermore, we found that MTDH protein expression negatively correlated with the TAX-containing chemotherapy efficacy (Fig. 1b). Based on these MTDH gene-associated clinical characteristics, we conducted the subsequent study to confirm the function of MTDH gene in breast cancer and its possible relationship with TAX chemotherapy.
Effect of MTDH expression on MCF-7 breast cancer cells
To investigate the effect of MTDH expression in breast cancer cell lines, we first performed RT-PCR tests on our modified MCF-7 breast cancer cells. We constructed four groups of shRNA and one control shRNA. After transfecting, amplifying, and extracting the four groups of plasmids, we selected the optimal silent shRNA (MTDH-shRNA3: 5′-GCAATTGGGTAGACGAAGAAA-3′) via RT-PCR and Western blot (Additional file 1: Figure S1). As shown in Fig. 2a, the relative MTDH mRNA expression level in our MCF-7–MTDH cell was 2.1 times higher than the reference MCF-7 cell. On the other hand, the relative MTDH mRNA expression level in our MCF-7–MTDH–shRNA cell was only 0.3 times the MCF-7 cell (Fig. 2b). Western blot tests were then conducted on MCF-7 cell and modified MCF-7 cells. We found that the protein level in MCF-7–MTDH cell was 2.86 times higher than the reference MCF-7 cell but in MCF-7–MTDH–shRNA cell was 90% lower than the reference cell (Fig. 2c, d). Furthermore, through CCK-8 assay, we observed that the growth rates of MCF-7–MTDH and MCF-7–MTDH–shRNA cells were higher and lower than that of the reference cell, respectively (Fig. 2e). Apart from that, through the cell apoptosis test, a test that measures the programmed cell death rate, we showed that the apoptosis percentage in an MCF-7–MTDH cell was much lower than MCF-7 cell but that of MCF-7–MTDH–shRNA was higher compared with MCF-7 cell (Fig. 2f, Additional file 1: Figure S2). In Fig. 2g and Additional file 1: Figure S4, we compared the proportion of G0/G1, S and G2/M phases in MCF-7–MTDH and MCF-7–MTDH–shRNA with the reference MCF-7 cell, respectively. Compared with MCF-7 cell, the MCF-7–MTDH cell had more S phase and less G0/G1 and G2/M phases while the MCF-7–MTDH–shRNA cell contained much lesser S phase and more G0/G1 and G2/M phases.
Relationship between MTDH expression and TAX treatment in MCF-7 cell
We now study how the MTDH expression in MCF-7 cell affects the effectiveness of the TAX drug treatment on breast cancer. We evaluated and compared the cytotoxicity of the modified MCF-7 cell with a reference MCF-7 cell through a CCK-8 assay. In Fig. 3a and b, we can see that the inhibition rate of the MCF-7–MTDH cell (25.89 ± 1.33%) was much lower than that of the MCF-7 cell (40.46 ± 1.31%) while that of MCF-7–MTDH–shRNA cell was the highest (64.33 ± 2.21%) at 48 h. In addition, the apoptosis percentage in MCF-7–MTDH cell (2.91 ± 0.89%) was lower than MCF-7 cell (9.31 ± 1.04%) while that of MCF-7–MTDH–shRNA cell (27.56 ± 2.40%) was higher compared with MCF-7 cell after TAX treatment (Fig. 3c, Additional file 1: Figure S3). As expected, the proportion of G2/M phase ratio in the MCF-7–MTDH cell was significantly lower than the MCF-7 reference cell after TAX treatment (Fig. 3d, Additional file 1: Figure S5). The NF-κB pathway was closely associated with chemotherapy resistance, so we examined the expression level of p65, p-p65, and IκBα. As shown in Fig. 3e, upregulation of MTDH increased p65 and p-p65 expression but reduced the expression of IκBα (suggesting the activation of NF-κB pathway). On the contrary, silencing of MTDH reduced p65 and p-p65 and increased IκBα expression. These results suggested that MTDH was related to NF-κB and TAX sensitivity.
Overexpression of MTDH promotes MCF-7 tumor growth in vivo and diminishes TAX activity
We further examined the effect of MTDH expression on in vivo MCF-7 tumor cell growth and TAX treatment efficiency using a mouse xenograft model. Figure 4a and b compared the in vivo images of xenograft tumors in untreated mice (MCF-7) and TAX-treated mice (MCF-7–MTDH and MCF-7–MTDH–shRNA). As can be seen, the tumor was significantly larger in the MCF-7–MTDH group but was smaller in the MCF-7–MTDH–shRNA group after subcutaneous injection of cells for 14 days. Mice were then treated with TAX by IP injection once a week for a total of four injections. The tumor in the MCF-7–MTDH–shRNA group was dramatically smaller than MCF-7–MTDH group as well as both controls (Fig. 4b), confirming that the knockdown of MTDH enhanced cell sensitivity to TAX exposure. Tumor volume measurement results also supported these observations (Fig. 4c, d). In addition, the tumor weight in the MCF-7–MTDH–shRNA group with or without TAX treatment was lower than that of the MCF-7–MTDH group or control group (Fig. 4e, f).
Development of MTDH–siRNA and TAX co-delivery technique
Based on the findings above, we have developed a new technique (amphiphilic copolymer PEI-PLGA) that allows both MTDH–siRNA and TAX to be concurrently delivered into breast cancer cells to effectively control the breast cancer condition. After emulsification twice, the TAX was loaded into the hydrophobic layer and the MTDH–siRNA was bound at the NP surface after addition through electrostatic interactions. Figure 5 shows the TEM images of blank NPs, TAX-encapsulated NPs (NP-TAX), and NP-TAX–siRNA. As can be seen, all NPs were dispersed with a well-defined spherical core shell structure. Dynamic light scattering (DLS) measurement suggested that the average hydrodynamic diameters of blank NPs, NP-TAX, and NP-TAX–siRNA were 218.5 ± 13.3 nm, 220.1 ± 9.1 nm, and 228.5 ± 10.4 nm, respectively (Fig. 5b, c). The zeta potentials of the blank NPs, NP-TAX, and NP-TAX–siRNA were 33.2 ± 0.6 mV, 42.4 ± 0.8 mV, and −22.5 ± 0.3 mV, respectively (Fig. 5c). When siRNA was mixed with NP-TAX, the zeta potential of NP-TAX–siRNA changed from 42.4 to −22.5 mV, indicating the successful and a large capacity of absorption of negatively charged nucleic acids on the NP surface. We also investigated the release profile of TAX at different pHs over time (Additional file 1: Figure S6). At pH 7.4, no significant release of TAX was observed in the first 10 h; however, TAX was released at a fast rate at pH 4.4, and a release ratio of around 40% was reached in the first 10 h. Therefore, the polymeric core is expected to show a pH-dependent drug release, which facilitates complete drug release in lysosomes after cellular uptake.
Cellular uptake and gene silencing of NP-TAX–siRNA
Two prerequisites for efficient siRNA-mediated gene silencing effect are high siRNA uptake levels and successful release of siRNA to cytoplasm. To study cellular uptake of NPs, we labeled NP-TAX–siRNA with near-infrared fluorescent dye Cy5. After incubation of MCF-7 cells with NP-TAX–Cy5–siRNA for 6 h, obvious red fluorescence appeared in the cytoplasm (Fig. 6a, upper panel). In contrast, there was barely red fluorescence in cells treated with free Cy5–siRNA (Fig. 6a, lower panel) and this was probably due to their high molecular weight, hydrophilic nature, and high density of charge. The knockdown efficiency of MTDH–siRNA encapsulated in NPs was then tested in MCF-7 cells by using RT-PCR and Western blot analysis. The downregulation of MTDH mRNA and protein expression in cells was observed after the NP-TAX–siRNA treatment, indicating the successful release of siRNA from lysosomes to cytoplasm (Fig. 6b, c). In addition, we examined the cytotoxicity of NP-TAX–siRNA to breast cancer cells in vitro. The MCF-7 cells were incubated with saline, blank NPs, free TAX, free siRNA, NP-TAX, NP-siRNA, or NP-TAX–siRNA for 48 h, followed by quantification of cell viability using a CCK-8 cell proliferation assay. As shown in Fig. 6d, compared with the saline group, free siRNA did not show significant inhibition in tumor cell growth. A possible reason for this phenomenon is that free siRNA cannot be taken up by cells easily. In contrast, the siRNA encapsulated in NPs (NP-siRNA) exhibits effective inhibition of cell growth, indicating successful delivery of siRNA into cells. Free TAX was more toxic than TAX encapsulated in NPs, while the inhibitory effect of NP-TAX–siRNA on cell growth outperformed all other groups. In addition, no cytotoxicity was observed in the blank NP-treated group, suggesting that the polymers are non-toxic. To confirm the inhibition effect of NP-TAX–siRNA on cell growth, we also tested cell proliferation after NP-TAX–siRNA treatment by using another human breast tumor cell line MDA-MB-435S (Fig. 6e), which has been demonstrated to express MTDH. Comparable results were observed.
In vivo biodistribution and antitumor activity of NP-TAX–siRNA
The MCF-7 cells were injected subcutaneously into BALB/c nude mice. When the tumors reached a size of about 100 mm3, Cy5.5-labeled NP-TAX–siRNA were injected by the tail vein. In vivo imaging results showed that the NPs gradually accumulated in tumor sites as a function of time (Additional file 1: Figure S7). Ten hours after the administration, we observe maximal fluorescence intensity in tumors, and at 24 h, the NPs were excreted from the mice body except for the tumor tissues, which is one of the desired characteristics of nanomaterials for in vivo application. In contrast, no special fluorescence was detected over time for the Cy5.5-labeled free siRNA treated group. We further performed ex vivo imaging assay. The mice were sacrificed after 10 h of administration, and the tumors and major organs (liver, heart, lung, spleen, and kidney) were collected. As can be seen in Fig. 7a, NP-TAX–siRNA accumulates mainly in the tumor tissues, and there is little accumulation in the liver and kidney and barely any in the heart, lung, and spleen.
Apart from that, we evaluated the antitumor activity of NP-TAX–siRNA using MCF-7 tumor-bearing mice. We randomly divided mice bearing about 100 mm3 tumors into seven groups: saline, blank NPs, free TAX, free siRNA, NP-TAX, NP-siRNA, and NP-TAX–siRNA. Mice were treated with different vehicles via the tail vein every two days for a total of 21 days. In the saline and blank NPs groups, the tumors grew fast and the mice were sacrificed two days after the last injection because of having large tumor size (~800 mm3) (Fig. 7b). Treatment with free siRNA or NP-siRNA has no significant inhibition effect on tumor growth. Although free TAX and NP-TAX slow down the tumor growth to a certain extent, their inhibitory effects are far lesser than the NP-TAX–siRNA.
To confirm the role of MTDH knockdown in the interference of tumor growth, we sectioned tumors and analyzed the levels of MTDH protein. Figure 7c shows that the MTDH expression level was dramatically suppressed by the NP-siRNA and NP-TAX–siRNA treatments compared with that in the saline group. Nevertheless, the free siRNA does not decrease the MTDH expression in tumor tissues. In the blank NP, free TAX, or NP-TAX–treated group, MTDH expression is similar to that in the saline group. In brief, the results suggest that siRNA is capable of effectively diminishing the MTDH gene expression in vivo only when delivered into the tumors by NPs and thus increasing the TAX antitumor effect.