*2.4. Annexin V-FITC/PI Assay Confirmed Apoptosis in MDA-MB-231 Treated Cells*

Annexin V-FITC/PI was employed to further confirm the mode of death in treated cells. As Figure 5 shows, apoptosis is induced in all treatments including 20 μM Gemini-Cur, 13 μM cisplatin and Gemini-Cur/Cis. However, the proportion of cells in late apoptosis is meaningfully increased to 59.3% in combination form compared to 15 and 42.6% in cisplatin and Gemini-Cur, respectively (*p* < 0.01).

**Figure 5.** The combination of cisplatin and Gemini-Cur induces breast cancer cell apoptosis. MDA-MB-231 cells were treated with 13 μM cisplatin, 20 μM Gemini-Cur, and Gemini-Cur/Cis for 48 h. (**A**): plots show that live cells undergo apoptosis and the number of death cells is increased in treated cells, especially in Gemini-Cur/Cis group. (**B**) The proportion of apoptotic cells in late stage is significantly increased in combination treatment. \*\*\*: *p* < 0.001.

#### *2.5. Expression of BAX, BCL-2 Genes Are Modulated in Treated Cells*

The expression ratio of BAX/BCL-2 is usually considered as a hallmark of apoptosis. Real-time PCR demonstrated that BAX/BCL-2 expression is modulated in treated MDA-MB-231 cells. As Figure 6 shows, apoptotic BAX is upregulated while anti apoptotic BCL-2 is downregulated in all treatments. Furthermore, this modulation was significant in Gemini-Cur/Cis treatments rather than cisplatin or Gemini-Cur groups. This differential effect of combination treatment was clearly detected in protein level (Figure 7A). Further analysis demonstrated that the protein ratio of BAX/BCL-2 is significantly increased in combination treatments (*p* value < 0.01) and Gemini-Cur (*p* value < 0.001) groups.

**Figure 6.** Quantitative analysis of the expression of apoptotic genes by real-time PCR. Relative expression for BAX (**A**) and BCL-2 (**B**) in MDA-MB-231 cells. Values represent mean ± standard deviation of three independent experiments; \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

**Figure 7.** (**A**) Western blotting for BCL-2 and BAX (**A**) proteins. β-actin was used as internal control. (**B**) Data analysis showed that the protein ratio of BAX/BCL-2 is significantly increased in the cells treated with Gemini-Cur/Cis compared to the groups of Cis and Gemini-Cur. Values represent mean ± standard deviation of three independent experiments. \*\*\* *p* < 0.001.

## **3. Discussion**

"Based on the St. Gallen/Vienna 2019 consensus discussion, 53% of the panelists didn't accept the use of a platinum-based regimen in the neoadjuvant treatment of TNBC patients" [19]. Presently, there are limited criteria for TNBC treatment, and what could help medical oncologists to cure TNBC or prolong the survival rate in TNBC patients are novel therapeutic strategies [20]. The present study aimed to sensitize TNBC MDA-MB-231 cells to cisplatin through employment of novel curcumin nano formulation (Gemini-Cur).

Our data showed that cisplatin modulates the proliferation of MDA-MB-231 cells in high concentrations. However, its combination with appropriate dose of Gemini-Cur not only reduced the IC50 value of cisplatin to 13 μM but also, boosted its suppressive effect on the growth of MDA-MB-23 cells. Due to the nephrotoxicity and hepatoxicity of cisplatin, decreasing its effective dose in cancer treatments is of interest in clinic [4,5]. Different therapeutic compounds have already been employed to improve the toxicity of cisplatin. More recently, it has been reported that curcumin reduces the resistance of colorectal cancer cells to cisplatin [21]. Here, the employment of Gemini-Cur, has significantly reduced the effective dose of cisplatin to 13 μM, a concentration that may not indicate side effects. Cell cycle analysis and microscopic visualization also demonstrated that the mode of death induced in treated-TNBC cells is of apoptosis.

Since the growth inhibitory effect of Gemini-Cur/Cis was more effective than singular treatments, it seems that Gemini-Cur increases the sensitivity of MDA-MB-231 cells to cisplatin. The anticancer activities of natural compounds such as curcumin has been examined in combination with numerous chemotherapy drugs such as cisplatin. It was observed that curcumin affects the toxic properties of cisplatin in different cancer cells [12,13,15]. It has also been shown that curcumin inhibits the function of P-gp efflux pump in cancer cells that might support the reversal effect of this phytochemical on therapeutic resistance of cancer cells [22].

Here, we indicated that the expression ratio of BAX/BCL-2 is modulated in apoptotic cells. These proteins are both essential gateways to cell death. Numerous studies have revealed that curcumin triggers apoptosis through BAX/BCL-2 mediated pathway in cancer cells [8,14]. The data is also in concordance with results of Karimpour et al. for the modulatory effect of Gemini-Cur on the expression of apoptotic genes in breast cancer cell lines [18]. The up regulation of BAX protein and down regulation of BCL2 was clearly detected in Gemini-Cur/Cis group compared to controls.

Taken together, the current results illustrate the synergistic property of Gemini-Cur as a novel nano formulation of curcumin on the therapeutic potential of cisplatin against drugresistant MDA-MB-231 cells. Enhancing the sensitivity of TNBC cells to cisplatin is still an interesting topic of cancer therapy for breast cancer. Therefore, it is worth investigating the exact cellular mechanisms and pathways involved in Gemini-Cur/Cis toxicity on TNCB cells. Furthermore, co-encapsulation of curcumin and cisplatin in gemini nanopartilces can be considered as an effective strategy to target cancer.

### **4. Materials and Methods**

#### *4.1. Reagents and Cell Culture*

Curcumin and mPEG urethane gemini surfactant nanoparticles were a kind gift from Dr. Farhood Najafi at the Institute for Color Science and Technology, Tehran, Iran. Cisplatin was obtained from Mylan Corporation, Germany. Gemini curcumin was formulated by dissolving 6 mg of curcumin and 100 mg of gemini nanoparticles in 3 mL methanol by using sonication at room temperature. Then, the organic phase was evaporated by a rotary evaporator in room temperature. The encapsulated nano curcumin was dissolved in 10 mL distilled water and stored at 4 ◦C for further analyses [11].

Human breast adenocarcinoma cell line, MDA-MB-231, was obtained from the national cell bank of Iran (Pasteur Institute, Tehran, Iran). The cells were cultured in DMEM-High glucose medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (Gibco, New York, NY, USA), and incubated at 37 ◦C in a humidified atmosphere of 5% CO2.
