*3.2. E*ff*ect of CA*/*siRNA*/*Drug at the Protein Level*

As cell viability assays revealed, silencing AKT or ERBB2 exerts the maximum effect on the enhancement of cytotoxicity of Pac or Doc on MCF-7. Thus, the underlying mechanism of reduced viability following administration of these treatments was investigated via western blotting. This was performed to confirm that the applied siRNA is effective in the knock down of the target protein synthesis and also to observe the changes in protein expression leading to enhanced chemo sensitivity.

AKT expression following treatment of MCF-7 cells with free AKT siRNA and CA-complexed AKT siRNA was assessed and compared with untreated cells. As the bands on the blot and densitometry analysis reveal (Figure 1), complexation of siRNA with carbonate apatite leads to significantly decreased expression of AKT compared to free AKT siRNA and untreated cells (*p* value < 0.05). This confirms the efficacy of CA in the successful delivery of siRNA and knock down of the target protein.

**Figure 1.** AKT protein expression in MCF-7 cells. Cells were treated with media (untreated), free AKT siRNA, and CA/AKT siRNA formed with 3 mM of CaCl2 and 1 nM siRNA for 44 h. Cellular lysates were resolved by SDS-PAGE and transferred in PVDF membrane followed by incubation with primary antibodies raised in rabbit against AKT. HRP-conjugated goat anti-rabbit secondary antibody was used to detect the chemiluminescent signals.

The same experiment design was utilized with MAPK siRNA. According to Figure 2, free MAPK siRNA does not change the expression level of MAPK protein whereas CA/MAPK significantly knocks down production of the target protein (*p* value < 0.05).

**Figure 2.** MAPK protein expression in MCF-7 cells. Cells were treated with media (untreated), free MAPK siRNA, and CA/MAPK siRNA formed with 3 mM of CaCl2 and 1 nM siRNA for 44 h. Cellular lysates were resolved by SDS-PAGE and transferred into PVDF membrane followed by incubation with primary antibodies raised in rabbit against AKT. HRP-conjugated goat anti-rabbit secondary antibody was used to detect the chemiluminescent signals.

Next, changes in protein expression resulting from the combinations of drugs and siRNAs, as highlighted in the cell viability data, were studied. MCF-7 cell lysate treated with free and CA-bound AKT siRNA, CA/AKT/Pac and CA/Pac were loaded on the gels. According to the resulting bands (Figure 3), the lowest level of AKT protein was obtained by CA/AKT/Pac treatment versus CA/Pac or CA/AKT. This implies the development of synergistic interactions to suppress survival pathways in the presence of classical anti-cancer drugs together with genetic downregulation of survival proteins, which in turn might lead to enhanced sensitivity of the cells to the anti-cancer medication.

**Figure 3.** AKT protein expression in MCF-7 cells. Cells were treated with media (untreated), carbonate apatite, free AKT siRNA, CA/AKT siRNA, CA/AKT/Pac, and CA/Pac. Ingredients include 3 mM of CaCl2, 1 nM siRNA, and 1 nM Pac. Cellular lysates were run in SDS-PAGE and transferred into PVDF membrane and incubated with primary antibodies raised in rabbit against AKT. HRP-conjugated goat anti-rabbit secondary antibody was used to detect the chemiluminescent signals.

The same synergistic pattern, but with a lower potency, was observed with application of CA/AKT/Doc in the downregulation of AKT protein against CA/AKT or CA/Doc (Figure 4).

**Figure 4.** AKT protein expression in MCF-7 cells. Cells were treated with media (untreated), carbonate apatite, free AKT siRNA, CA/AKT siRNA, CA/AKT/Doc, and CA/Doc. Ingredients include 3 mM of CaCl2, 1 nM siRNA, and 1 nM Doc. Cellular lysates were run in SDS-PAGE and transferred into PVDF membrane and incubated with primary antibodies raised in rabbit against AKT. HRP-conjugated goat anti-rabbit secondary antibody was used to detect the chemiluminescent signals.

Based on a substantial enhancement in efficacy, the impact of different combinations of ERBB2 siRNA with paclitaxel or docetaxel on the expression level of ERBB2 protein was also examined. As the blot in Figure 5 shows, the highest knock down was achieved by co-delivery of CA-bound Doc and ERBB2 siRNA followed by CA/Pac/ERBB2. Again, the synergy in the therapeutic efficacy of the drug together with siRNA is present since the impact of the drug or siRNA as a single agent is considerably weaker.

**Figure 5.** ERBB2 protein expression in MCF-7 cells. Cells were treated with media (untreated), carbonate apatite, free ERBB2 siRNA, CA/ERBB2 siRNA, CA/Doc/ERBB2, CA/Pac/ERBB2, CA/Doc, and CA/Pac. Ingredients include 3 mM of CaCl2, 1 nM siRNA, and 1 nM drug. Cellular lysates were run in SDS-PAGE and transferred into PVDF membrane and incubated with primary antibodies raised in rabbit against ERBB2. HRP-conjugated goat anti-rabbit secondary antibody was used to detect the chemiluminescent signals.

#### *3.3. In Vivo E*ffi*cacy of CA*/*Drug*/*siRNA*

According to cell viability data on 4T1 cells and the enhancement in the cytotoxicity of drugs, co-delivery of AKT and ERBB2 siRNAs together with Pac or Doc resulted in the highest increase in the drug's efficacy. Thus, these two combinations were applied in animal study.

For comparison of the in vivo efficacy of the therapeutics in the first batch, animals were treated on day 8 and 11. Formulations were prepared by mixing 4 mM Ca, 1.25 mg/kg Pac, 50 nM AKT siRNA plus 50 nM ERBB2 siRNA in 100 μL of HCO3-DMEM. There were no significant changes in the pattern of body weight change and also general signs and symptoms of the animals among different groups of the studies.

As the t-test results reveal, CA-AKT-ERBB2-Pac treatment significantly reduced the tumor volume on day 14 and 16 compared to Ca/Pac. Moreover, the group treated with CA/ERBB2 displayed significantly smaller tumors compared to the CA group on day 12 and 14. The effect of Pac complexed with CA on tumor regression was significant on day 14 versus free paclitaxel therapy (Figure 6).

**Figure 6.** Effect of silencing AKT and ERBB2 pathways on in vivo efficacy of paclitaxel. Mice were purchased from Razi Research Institute, Tehran, Iran. Approximately 106 4T1 cells were inoculated subcutaneously on the mammary pad of mice. Based on tumor volume calculations, mice were randomized and treated intravenously through tail-vein injection on day 8 and 11. The therapeutics included 100 μL of carbonate apatite entailing 4 μL of 1M CaCl2 and 50 nM of AKT and ERBB2 siRNA plus 1.25 mg/kg Pac. Body weight and tumor outgrowth were monitored every other day. Data is represented as mean ± SD, *n* = 6 and values are significant when \* *p* value < 0.05 for CA/Pac/AKT/ERBB2 vs. CA/Pac, # *p* value < 0.05 for CA/ERBB2 compared to CA and \*\* *p* value < 0.05 for CA/Pac against Pac.

In another batch, injections on day 10 and 13 encompassed preparations of 4 mM Ca, 1 mg/kg Doc, 50 nM AKT siRNA, and 50 nM ERBB2 siRNA in 100 μL bicarbonated DMEM (Figure 7).

**Figure 7.** Effect of silencing AKT and ERBB2 pathways on in vivo efficacy of docetaxel. Mice were purchased from Razi Research Institute, Tehran, Iran. Approximately 106 4T1 cells were inoculated subcutaneously on the mammary pad of mice. Based on tumor volume calculations, mice were randomized and treated intravenously through tail-vein injection on day 10 and 13. The therapeutics included 100 μL of carbonate apatite entailing 4 μL of 1M CaCl2 and 50 nM of AKT and ERBB2 siRNA plus 1mg/kg Doc. Body weight and tumor outgrowth were monitored every other day. Data is represented as mean ± SD, *n* = 6 and values are significant when \* *p* value < 0.05 for CA/Doc/AKT/ERBB2 vs CA/Doc, # *p* value < 0.05 for CA/ERBB2 compared to CA and \*\* *p* value < 0.05 for Ca/Doc against Doc.

According to the *p* values calculated in the t-test, treatment of animals with CA/Doc/AKT/ERBB2 resulted in significantly smaller tumor volumes on days 16, 18, 20, and 22 compared to CA/Doc. Whereas CA/ERBB2 displayed a significant anti-tumor efficacy only on day 16 compared to CA, and CA/AKT was not effective in tumor regression. Animals treated with CA/Doc had significantly smaller tumors on day 18 and 20 compared to Doc-treated animals.

ERBB2 overexpression has been linked to elevated levels of inhibitory phosphorylation of Cdc2 and suppression of paclitaxel-induced cell death in breast cancer cells via deregulation of the G2/M cell cycle checkpoint. This provides a mechanistic rationale for the association between ERBB2 overexpression and paclitaxel resistance. Interestingly, overexpression of a subunit of PI3k in ovarian cancer cells has been shown to confer paclitaxel resistance. Additionally, selective inhibition of the PI3k pathway could restore the efficacy of paclitaxel in those cells [25]. Thus, alterations in the expression and activity levels of key components of these signaling networks regulating cellular proliferation and survival may confer paclitaxel resistance. Further, circumvention of this type of resistance might be achieved via application of selective inhibitors of these proteins to increase drug sensitivity.

Simultaneous targeting of AKT and ERBB2 to achieve improvements in eradication of oncogenesis has been brought up in various studies.

Overexpression or activation of HER members and also AKT has been linked to limited benefits of treatment in patients, leading to poor prognosis in breast cancer. In fact, functionally relevant alterations in AKT1 could be a putative mediator of tumor progression and drug resistance. Moreover, amplification or gain-of-function mutations of ERBB2 can account for hyperactivation of the AKT cascade in breast cancer cells. Crosstalk with heterologous receptors and amplification of HER2 signaling, amplifications of the PI3K/AKT pathway, and de-repression and/or activation of compensatory survival pathways through increased PI3K/AKT signaling are among the resistance mechanisms against anti-HER2 therapy. Factors associated with resistance to ERBB2-targeted agents have been invariably associated with a reactivation of the PI3K/AKT signaling cascade [26,27]. However, defects in HER2/PI3K/AKT axes and their impact on regulation of the response of cancer cells to treatment need further investigation.

In an experimental design, HER2-positive MCF7 cells showed a PI3K-dependent increase in AKT activity together with increased resistance to a panel of five chemotherapeutic agents with known different mechanisms of action (paclitaxel, doxorubicin, 5-fluorouracil, etoposide, and camptothecin). Selective inhibition of PI3K or AKT in these HER2-overexpressing MCF7 cells reduced the levels of phosphorylated (activated) AKT and sensitized the cells to the chemotherapeutic agents. It has been further confirmed that expression of a constitutively active AKT vector alone in MCF7 cells caused similarly increased resistance of the cells to the chemotherapeutic agents [13]. Therefore, the HER2/PI3K/AKT pathway is confirmed to play a causal role in the resistance of breast cancer cells against several therapeutics, and targeting components of this pathway might resolve the resistance and enhance the efficacy of the treatments. In view of that, the in vitro and in vivo results of this study are in alignment with the documented role of AKT and ERBB2 in drug resistance.

Strikingly, the efficacy of simultaneous knock down of AKT and ERBB2 in augmenting the response of cancer cells to docetaxel was reported for the first time in this study.

Markedly, the strategic location of AKT and its activation by multiple upstream signal transduction pathways makes it a better target than its upstream targets, such as HER2, Ras, or PI3K, in sensitizing cancer cells to chemotherapy or radiotherapy. Thus, there could be clinical benefits from an appropriate combination of conventional chemotherapeutic drugs with a new generation of signal transduction inhibitors that inhibit the HER/PI3K/AKT pathway for the treatment of breast cancer.

A summary of the ERBB2 and AKT signaling cascades regulating cell's proliferation, survival, and resistance to apoptosis and treatment is illustrated in Figure 8. This is for clarification of the enhanced efficacy of Pac or Doc attained by simultaneous blockade of AKT and ERBb2 cascades and the impact on the response of the cells to the drugs.

**Figure 8.** Summary of the ERBB2 and AKT signaling cascades regulating the cell's proliferation, survival, and resistance to apoptosis and treatment.

As revealed through the extensive experiments on breast cancer cells and animal models plus protein expression studies, simultaneous knock down of AKT1 and ERBB2 expression in the presence of paclitaxel or docetaxel leads to a substantial increase in cellular response to the chemotherapeutic agent both in culture and an animal model. Synergistic interactions in target protein knock down have been documented via western blotting in the cells treated with these combinations, which would shed light on the underlying mechanisms of the enhanced sensitivity of the cells to treatment. Taken together, with a favorable in vitro profile and also superior in vivo efficacy, co-administration of genetic materials and classical chemotherapeutics could propose a promising platform for improved strategies against cancer cells in upcoming practice.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/11/9/458/s1, Figure S1. Cell viability assessment in 4T1 cells treated with siRNA loaded carbonate apatite. Figure S2. Carbonate apatite facilitated delivery of ERBB2 (A) and ROS1 (B) siRNA to MCF-7 cells. Figure S3. Effect of single pathway silencing on cytotoxicity of paclitaxel on 4T1 cells. Figure S4. Effect of single pathway silencing on cytotoxicity of docetaxel on 4T1 cells. Figure S5. Effect of single pathway silencing on cytotoxicity of mitomycin C on 4T1 cells. Figure S6. Effect of single pathway silencing on cytotoxicity of topotecan on 4T1 cells. Figure S7. Effects of single pathway silencing on paclitaxel cytotoxicity in MDA-MB-231. Figure S8. Effects of single pathway silencing on docetaxel cytotoxicity. Figure S9. Effects of silencing AKT and ERBB2 oncogenes on drugs cytotoxicity in 4T1. Figure S10. Cell viability assay on MDA-MB-231 cells treated with carbonate apatite complexed with paclitaxel and two siRNAs.

**Author Contributions:** T.F. carried out experiments and data analysis under the supervision of H.R.M. and E.H.C., H.R.M. provided resources while E.H.C. originally designed the project and provided the resources.

**Funding:** This research was supported by MOSTI Science fund (Project ID: 02-02-10-SF0299) of Malaysia.

**Conflicts of Interest:** The authors declare no conflict of interest.
