At present, the incidence of lung cancer in China has increased significantly, and has become the highest mortality cancer [16]. Cisplatin is being used extensively to treat various cancers, especially lung cancer [17]. However, its virulence and desensitization are concerns. Research is ongoing to overcome its toxicity and to ameliorate cisplatin sensitivity. Although decreasing the cisplatin dosage reduces the toxicity, its antitumor activity is also attenuated. Therefore, to find a new combination chemotherapy regimen with reduced toxicity and high efficiency, has become an urgent issue. Dihydromyricetin (DMY] is a natural flavonoid extracted from Ampelopsis grossedentata and Myrica rubra, which has been proved to have strong antitumor activity, but little effect on normal cells in various human carcinoma cell lines [18, 19].
It has been reported that DMY could reverse human gastric cancer SGC7901 / Adr cells multidrug resistance [12]. In addition, the synergistic antitumor effect of DMY combined with other drugs was also evaluated by enhanced efficacy and reduced side effects [20–22]. Consistent with the above reports, our results demonstrated that DMY and cisplatin show strong cooperative effects in inhibiting the proliferation, migration, invasion and inducing apoptosis of lung cancer cells.
In this work, MTT assay was used to test the inhibitory effects of A549 cells. Single use of cisplatin or DMY was able to induce growth inhibition, furthermore, the inhibitory effect of combined drugs was better than that of two single agents. Based on the term CI proposed by Chou and Talalay in 1984 [23], which is quantification of synergism or antagonism effect of two combination drugs. We demonstrated that DMY synergized with cisplatin in various combinatorial drugs concentration, with a mean CI all less than 1. Under this synergistic effect, the combined treatment of DMY and cisplatin could strengthen the antitumor effect of cisplatin, decrease toxicity of cisplatin, and retard the acquisition of cisplatin resistance that possibly occur.
Next, we evaluated the effect of a combination regimen of low dose DMY and low dose cisplatin on A549 cells. In the results of the apoptosis study, it was found that neither low-dose DMY nor low-dose cisplatin alone had a significant effect, but the combination treatment group significantly increased apoptosis. The combination of the two drugs had a synergistic effect, achieving a therapeutic effect and reducing the amount of chemotherapy drugs used. The same results were seen in studies of invasion and migration.
The following studies explore the mechanisms. MMPs are proteases that play an important role in cancer migration and invasion, degrading the basement membrane and extracellular matrix and disrupting the cancer cell invasion barrier. Based on substrate and fragment homology, MMPs are divided into six major classes, namely collagenase, gelatinase, matrix degradation, matrix lysine, furan-activated MMP and other secreted MMP. MMP 2 and MMP 9 are the key components of MMP family. MMP 2, also known as gelatinase A, hydrolyses collagen types IV, V, I and III, laminin and elastin, which is widely distributed in vivo, being expressed in most cells including stromal cells, endothelial cells and epithelial cells [24]. MMP 9, also known as gelatinase B, hydrolyses various components of the basement membrane and extracellular matrix, such as collagen IV, and thus plays a key role in cancer cell migration and invasion [25]. High expression of these two proteins is essential in the invasion of a wide range of cancer cells. In this study, the protein expressions of MMP 2 and MMP 9 were significantly down-regulated in the combined group, which might be an potential mechanism that DMY and cisplatin synergistically inhibited the migration and invasion of A549 cells [26].
As we know, migration and invasion of cancer cells are often accompanied by the process of EMT. During this process, the expression of e-cadherin, a phenotypic marker protein, is gradually lost in epithelial cells, while n-cadherin, a stromal marker protein, is increased. EMT down-regulated the intercellular adhesion ability of lung cancer cells and upregulated the migration and invasion ability, then result in the metastasis of lung cancer [27, 28]. Previous studies suggested that DMY suppressed tumor growth and EMT through regulating miR-455-3p in human cholangiocarcinoma [29]. Our research showed that DMY alone or combined with cisplatin up-regulated the expression of epithelial marker E-cadherin protein, and down-regulated the expression of mesenchymal marker N-cadherin. These results indicating the EMT of lung cancer cells was inhibited by DMY. Previous report suggested that SMAD 3, SMAD 4-mediated EMT may promote migration and invasion, thereby linking it to poor prognosis and chemoresistance in NSCLC [30]. While our results found that the combination of DMY and cis could synergistically decrease the level of p-SMAD 3, t-SMAD 3 and t-SMAD 4, suggesting that DMY might enhance the inhibition of cisplatin on the EMT of A549 cells by decreasing SMAD 3 / SMAD 4 protein expression levels or decreasing SMAD 3 phosphorylation levels.
In conclusion, DMY enhanced the inhibitory effect of cisplatin on A549 cell proliferation, invasion and metastasis, and the mechanism may be related to the influence of the expression level of some related proteins including MMP 2, MMP 9, E-cadherin, N-cadherin, p-SMAD 3, t-SMAD 3 and t-SMAD 4. These results will provide new ideas and methods for the clinical treatment of lung cancer.