Changes in HER2 function are a common molecular defect in breast malignancy and commonly associated with an aggressive phenotype and poor outcomes [2, 3]. Clinically, two main problems may be occurring after using HER2 inhibitors in breast tumors with HER2 overexpressing. First, the effect of HER2 inhibitors is limited by the development of hereditary and acquired resistance that usually occurs within one year after starting the therapy [4]. Second, since the activity of HER2 has a vital role in heart function and development, thus, the risk of cardiomyopathy should be considered [22, 23]. Though, proposed that simultaneous HER2 inhibition and AMPK activation, may demonstrate very success in cancer treatment, and ameliorate cardiac side effects [24]. Moreover, several studies indicated that combination chemotherapy had a high ability to reduce the growth of carcinoma cells, prevent angiogenesis, induction of apoptosis, and triggers an immune response [8–10]. Recently, combination therapy of chemotherapeutic agents and phytochemicals is more attention as a successful alternative therapy to increase therapeutic efficacy and decreases the systemic toxic effects of these chemotherapeutic agents. Although, various in-vitro and in-vivo combination studies between MET and chemotherapeutic agents were performed to analyze the effects of such combination on the inhibition of cell proliferation, cell cycle regulation, and apoptotic death. However, to the best of our knowledge, the combination of MET and LAP has not been examined, therefore, in the current study; we assessed the effect of MET and LAP either individually or in combination on the cell viability and apoptotic death in SKBR3 cells.
We found that LAP and MET individually and in combination had a significant toxic effect on the SKBR3 cells in a time and dose-dependent manner. Analysis of flow cytometry data showed that the MET-LAP combination had a more apoptotic death compared to both of them individually. Also, the combination could significantly increase the mRNA expression of pro-apoptotic protein Bax, a cell-cycle inhibitory protein, p21, and caspase 3 and 9 activity. Whereas, the mRNA expression of anti-apoptotic protein Bcl2 was significantly decreased. Moreover, the combination could significantly increase the AMPK and decrease the Akt expression. Our results are in accordance with the results from preceding studies which confirmed the inhibitory effect of MET [17, 25, 26] and LAP [10, 27, 28] on the breast cancer cell lines. Moreover, they found a combination of LAP and MET could considerably reduce the growth of all cell types compared to the treatments with them individually.
Clinically, MET is commonly safe, worldwide available and it is significantly low-cost. Although, it is the first-choice oral therapy as an anti-diabetic drug, several in vitro and in vivo studies have shown that MET has anti-cancer activity in several malignancies, such as pancreatic, prostate, colorectal, and breast cancers [14–17]. Several mechanisms have been suggested to explain such anti-cancer activities of MET, for example, activation of AMPK and inhibition of mTOR [15, 29]. It is well established that Akt has a key role in the proliferation of carcinoma cells and apoptotic death. Because, the Akt is downstream of several coordinate tumor growth factors including IGF- I receptor [20, 31]. Thus, our finding that the MET-LAP combination reduced the Akt expression in SKBR3 cells may represent an important effect of this drug for controlling carcinoma cell proliferation.
A case-control study by Jiralerspong et al. [32] indicated that diabetic patients with breast cancer treated with neoadjuvant chemotherapy taking also MET had a higher pathologic response compared with patients not taking. In a study by Oliveira-Ferraro et al. [33], they found MET can inhibit the expression of M phase-related genes in the breast cancer cells by activating the AMPK pathway, consequently, the breast cancer cell cycle was arrested in the G2 phase. A study conducted by Sahra et al. [14] indicated, although, MET could inhibit the growth of the prostate cancer cells, however, could not induce apoptotic death. Other studies showed that MET can induce apoptotic death in breast, colon, and endometrial carcinoma cells [34–36]. Zhuang and Miskimins [36] found that MET individually, or in combination with tamoxifen could increase apoptotic death in SKBR3cells via caspase and PARP activity. Also, they showed that MET could increase cell apoptosis via the activation a caspase-mediated pathway. These results are consistent with our result that found MET could increase the caspase 3and 9 activity in combination with LAP.
LAP, a dual synthetic reversible inhibitor of tyrosine kinase activity, has potent anti-tumor activity against HER2-overexpressing breast cancer [9–12], but the exact mechanism of its anti-tumor action has not been completely understood. Akt/PKB is a key serine/threonine kinase that regulates several cellular signaling pathways including cell proliferation, viability, size, and invasion, together with glucose metabolism and angiogenesis [17]. Our results indicated that although MET and LAP individually could not affect the expression of AMPK and Akt. However, the MET-LAP combination could increase and decrease the expression of AMPK and Akt, respectively. Previous studies reported that MET could inhibit signal transduction through ERK and Akt, both of which advocate cancer development in several malignancies such as breast cancer [37, 38]. The alteration of Akt function is usually dis-regulated in various types of malignancy and has a vital role in tumor cell invasion, proliferation, mobility, and survivance [38]. The regulation of apoptosis by Akt may be mediated through phosphorylation of several pro-apoptotic proteins including Bax and caspase 9 [39]. Our results indicated that MET and LAP individually and in combination could significantly increase the expression of Bax and caspase 3 and 9 activity in SKBR3 cells. It is speculated that these effects may be mediated by the reduction of protein level of Akt. Moreover, Akt can induce phosphorylation and inactivation of the Forkhead Box (FOXO), which is implicated in the expression of pro-apoptotic genes. On the other hand, Akt can phosphorylate and inactivate IκB kinase (IKK), a positive modulator of NF-κB that induce several anti-apoptotic gene expressions [40]. Also, phosphorylation of Mdm2 by Akt resulted in its activation and induces its movement in the nucleus, where it promotes the degradation of p53 [41, 42]. Otherwise, Akt by induction of several transcription factors phosphorylation, can decrease p53 mRNA expression [43]. Our results indicated that MET and LAP individually and in combination could significantly increase the expression of a cell-cycle inhibitory protein, p21, which is a target of P53. It is suggested that this effect may be due to the reduction of the inhibitory effect of Akt on the P53 expression.
Since inhibition of HER2 tyrosine kinase activity is associated with blocking of downstream signaling pathway of Akt and AMPK in HER2-overexpressing breast carcinoma cells [44], thus, to gain more insight into the LAP-induced apoptotic death, its molecular docking studies were performed on the Akt, AMPK, and HER2 targets. Results of the molecular docking verified the inhibitory effect of LAP on the HER2. LAP showed an interaction energy value (∆G bind) of -11.31, -7.54, and − 11.66 kcal.mol− 1 in binding to Akt, AMPK, and HER2, respectively. However, it showed stronger binding energy in binding to HER2 in comparison to Neratinib (-9.13 kcal.mol− 1), as HER2 tyrosine kinases inhibitors. We confirmed that the main amino acids inside the active site of the HER2, which is responsible for essential interactions with LAP are Leu718, Phe723, Val726, Ala743, Lys745, Thr790, Leu792, Met793, Leu844, Asp855, and Phe856. Immunoblot analysis showed that MET-LAP combination could decrease and increase the expression level of Akt, and AMPK. Which is in consistent with the molecular docking results, which showed that the inhibitory effect of LAP in binding to HER2 can down-regulate and up-regulate the Akt and AMPK, respectively. Molecular docking of LAP in binding to Akt, and AMPK was also conducted to find out the binding modes, binding sites, and the best binding orientation of LAP to Akt and AMPK. Even though the binding of LAP to Akt, and AMPK cannot exactly explain the mechanism of down-regulation, and up-regulation of Akt, and AMPK, respectively.