Despite its therapeutic significance in multiple human cancers, curcumin-based treatments suffer some drawbacks, including its inadequate bioavailability, low-aqueous solubility, and poor absorption, demanding new strategies for improvement of the cytotoxic effects of curcumin . Subsequently, nowadays, biodegradable polymeric nanomaterials, because of some advantages such as bioavailability, stability, and controlled release are suggested to develop new delivery systems and improve effectiveness of curcumin as a hydrophobic component . Therefore, the current study was aimed to examine the improvement of curcumin on breast cancer growth and metastasis trough loading on Gemini surfactant NPs.
The obtained results from in vitro analysis illustrated that loading of curcumin on Gemini NPs increased not only the cellular uptake but also inhibitory effect of curcumin on 4t1 breast cancer cell growth and proliferation. Further in vitro analysis illustrated that Gemini-Cur also led to a reduction in tumor incidence, size and weight in breast cancer models. To understand the mechanism underlying anti-proliferative effect of Gemini-Cur, flow cytometry analysis was performed. Annexin V/PI staining results implied that Gemini-Cur inhibited in vitro and in vivo breast cancer growth through apoptosis induction. Moreover, qRT-PCR results demonstrated that Gemini-Cur induces apoptosis in breast cancer cells through modulating the expression of important member of the Bcl-2 family genes, including Bax and Bcl-2 expression. Deregulated expression of these genes blockades the programmed cell death and promotes tumor growth. Subsequently, Bcl-2 expression level, as an important pro-survival gene that prevents cells from apoptosis induction, was downregulated in treatment group compared to the control, while pro-apoptotic gene of Bax that is involved in release of cytochrome c activating caspase cascade during apoptosis, showed higher expression levels in comparison with control.
Metastasis is considered the leading cause of breast cancer mortality. This process is consequence of tumor cell ability to invade and migrate to other organs. In particular, 70–80 percent of breast cancer cases suffers from invasive form malignancy having potential to metastasize to neighboring and distance body organs through the bloodstream and lymphatic system, which is main cause of breast cancer related deaths. Considering that, in the current study, the effectiveness of designed NPs on anti-metastasis effects of curcumin was also investigated. In vivo histological results established that appearance of invasive tumors in treatment groups was remarkably lower than the control group. To further confirm anti-metastasis effect of Gemini-Cur on breast cancer, the expression of MMP9 was also measured through the study. qRT-PCR showed that treatment with Gemini-Cur led to a reduction in MMP9 expression in BC tumors. MMP9, as an important matrix metalloprotease, is upregulated through breast tumorigenesis which is correlated with poor survival of patients. MMP9 is capable to degrade basement membrane collagens, leading tumor cells to invade, migrate and metastasize. Consistent with our results, Na Mo and colleagues have previously reported that exposure to curcumin inhibited breast cancer cell invasion through downregulation of TGF-β1-stimulated MMP-9 expression . Furthermore, it was illustrated that endosomal curcumin could reduce MMP9 expression and inhibit invasive tumor appearance in breast cancer mice models . Therefore, it was suggested that Gemini-Cur could also show anti-metastasis effect on breast cancer cells through regulating MMP9 expression.
Angiogenesis through the formation of new vascular networks is a key mechanism to accomplish excessive need of tumor cells for oxygen and nutrients, supporting their rapid and uncontrolled growth and proliferation . Consequently, the expression levels of Vascular endothelial growth factor A (VEGFA), as important modulator of angiogenesis were investigated in the present study. VEGFA is upregulated through tumorigenesis of multiple cancers, including breast cancer which is correlated with aggressive from of malignancy . Besides, VEGFA has been illustrated to provoke stemness, invasion and metastasis in breast cancer cells through modulating mesenchymal markers, including fibronectin, vimentin and N-cadherin . Therefore, targeting VEGFA, as an angiogenic factor involving in initiation and progression of tumor neovascularization is considered a promising option for treatment of several malignancies such as breast cancer . Interestingly, we also found that treatment of breast cancer mice models with Gemini-Cur reduces VEGFA expression in comparison with controls, indicating the significance of designed Curcumin loaded-NPs in suppression of angiogenesis as well. To support this hypothesis, Thulasiraman and colleagues established that curcumin, in addition to increasing the sensitivity of triple negative breast cancer cells to retinoic acid, could reduce VEGFA expression levels . Moreover, curcumin was shown to induce apoptosis in vitro and to suppress in vivo tumor growth and angiogenesis in breast cancer models via modulating NF-κB signaling pathway which is involved in the regulation of VEGF expression [26, 27]. Therefore, it could be concluded that Gemini-Cur may also exert suppressive effects on angiogenesis of breast cancer cells.
Finally, the effect of Gemini-Cur on Cyclooxygenase 2 (COX-2) expression, as an important factor participating in human breast cancer angiogenesis and lymph node metastasis , was examined in treatment groups. The results confirmed that treatment of breast cancer models with Gemini-Cur could effectively downregulate COX-2 expression. COX-2 was previously established to play important part in VEGF-induced angiogenesis through activation of JNK and p38 kinase pathways in vascular endothelial cells. Also, Ki Won Lee and colleagues reported that in human breast epithelial cells curcumin could suppress COX-2 and MMP-9 via inhibition of ERK1/2 phosphorylation and trans-activation of NF-kB . Furthermore, Curcumin has been shown to repress the development of several types of human malignancies by decreasing the production of inflammatory mediators, including COX-2 . Therefore, considering the aforesaid evidences, Gemini-Cur was suggested to induce its suppressive effects on migration and angiogenesis through downregulating COX-2 expression, as a major regulator participating in tumor development.
In conclusion, the results of current study illustrated that loading of curcumin on Gemini surfactants NPs increased its anti-proliferative effect on breast cancer cells through apoptosis induction. This effect was accomplished by modulating the expression of major cell survival regulators including, Bax and Bcl-2. Furthermore, the obtained results showed that Gemini-Cur could effectively inhibit tumor growth in breast cancer mice models. Besides, suppressive effect of Gemini-Cur on metastasis and angiogenesis via regulating MMP9, VEFGA and COX-2 expression was illustrated through current study. Therefore, it is suggested that Gemini surfactants NPs could be considered as an effective strategy for better delivery of curcumin, improving its anti-tumor effects on breast cancer cells.