Selective cytotoxicity of a Vietnamese traditional formula, Nam Dia long, against MCF-7 cells by synergistic effects

Cancer is a world-wide health issue, with growing incidence due to increased industrialization and changes of environmental conditions, including lifestyle. Sophisticated methods based on chromatographic separation are used for isolation of pure anticancer substances from natural sources. On the other hand, combinatorial chemistry generates numerous synthetic chemicals for anticancer purposes. However, only a few clinically significant chemotherapeutic agents were discovered through large screening projects, and the development of synthetic drugs that have both efficacy and low toxicity were relatively inefficient, though extremely costly [16]. A totally different approach, represented by various traditional medicine systems, including VTM, assumes that maximal therapeutic efficacy can be obtained through complicated interactions among all ingredients of a formulation. These interactions can be synergistic, additive or antagonistic to each other aiming at reducing adverse side-effects, or enhancing and prolonging the therapeutic process [1, 17]. Since cancer is a multifactorial and highly heterogeneous disease, polychemotherapy, such as multi-ingredient formulations, is considered to be more active than single agent treatment [2, 17]. In this study, NDL and its four ingredients were prepared as aqueous extracts, since in VTM the most frequent administration form is aqueous decoction. We used the median-effect principle of Chou and Talalay to quantify the effect of combined ingredients from NDL. The advantage of using this method was that the determination of synergism, antagonism or additive effects does not require an understanding of the mechanism of action of the complete formula or its separate ingredients [15]. NDL exhibited significantly higher activity compared to the separate ingredients. Cell growth inhibition by NDL and the four combinations, each lacking one ingredient, showed that all ingredients were necessary for the overall cytotoxic effect, even though they possessed cytotoxicity at different levels. Sweet leaf (Sa) seemed to be the leading component for cytotoxicity of NDL, in agreement with previous reports showing extracts from S. androgynus induced apoptosis and necrosis on fibroblasts and endothelial cells [10, 11]. Earthworm (Pa), with its low cytotoxicity, possibly contributed through another activity. CI values calculated for NDL concentrations of IC₃₀ to IC₆₀ values were less than 1, indicating strong and stable synergistic interactions among all ingredients. At the high concentration of the IC₈₀, the NDL ingredients showed antagonistic interactions. Real-time monitoring of cell growth showed that MCF-7 cells treated with NDL exhibited a clear cytostatic profile, different from control cells, for more than 20 h. During this period, the four ingredients alone and three-ingredient combinations shared a kinetic profile similar to the control cells. This suggested a different mode of action of NDL other than what is exerted by the single ingredients and the three-ingredient combinations. As previously reported, biological activities of a herbal combination resulted from interactions among the different components rather than from activities of individual ingredients [18]. Moreover, the mode of action of a herbal formula could be more or less different from individual ingredients, expressed by different profiles of gene expression of treated cells [19].

In chemotherapy, high cytotoxicity toward cancer cells with minimal harm to normal cells is the ideal approach. The selectivity index (SI) reflects the differential cytotoxicity of a compound against tumor and normal cells. The greater the SI value of a compound, the more selective it is. An SI value above 2 indicates cytotoxic selectivity [20]. At the IC₅₀ concentration, NDL cytotoxicity was significantly greater on MCF-7 and Hep G2 cells than on normal fibroblasts. NDL also exhibited higher cytotoxicity against NCI-H460 than fibroblasts, though the difference was not significant. The SI values of NDL, calculated from the cytotoxicity of NDL against normal fibroblasts and MCF-7, Hep-G2 and NCI-H460 were 6.45, 1.61, and 1.29, respectively, indicating a highly selective cytotoxic effect of NDL against MCF-7 cells and a general toxicity to Hep-G2 and NCI-H460 cells. The high cytotoxicity of NDL toward MCF-7 cells could be explained partly by the presence of flavonoids in the NDL ingredients (data not shown). The cytotoxicity of flavonoids on breast cancer cells, such as MCF-7 cells, is closely related to the expression of estrogen receptors [21]. In an ongoing study, we showed that NDL upregulated the expression of estrogen receptor beta (data not published), which was shown to inhibit human mammary epithelial cell growth [22]. Real-time monitoring of cell growth in the short term revealed a rapid cell index increase only on MCF-7 cells after treatment with NDL at a concentration of 4.3 mg/mL followed by rapid cell index decrease, corresponding to cell detachment. This kinetic profile resembled the profile generated by thapsigargin, a compound that modulates intracellular calcium level [23]. The increase of cytosolic calcium leads to transient changes in cell morphology, which explains the change of cell index value [24]. Polyphenolic compounds induce calcium release and disrupt mitochondrial function, leading to selective cytotoxicity toward MCF-7 cells [25]. Long-term monitoring of MCF-7 cell and fibroblast proliferation showed that they responded differently to the same concentrations of NDL. Fibroblasts maintained a better and longer survival rate compared to tumor cells. The antiproliferative effect of NDL was expressed through a cell cycle non-phase-specific as indicated by flow cytometry analysis. When cells were induced to a cytostatic status, they should resolve the situation either by death or by escape from the growth inhibitory pressure [26]. Treated fibroblasts can escape the cytostatic effect of NDL, but MCF-7 cells cannot and underwent apoptosis as a response to NDL treatment. Bioactive substances from each ingredient may contribute directly to the overall effect. Naringin, a flavonoid from mung bean, was reported to inhibit P-gp and breast cancer resistant protein, thus improving drug absorption [27]. Quercetin, found in mung bean seeds and sprouts, was shown to upregulate estrogen-binding sites type-II, thus exerting an inhibitory effect on MCF-7 cells even at low concentration [28]. Interestingly, naringenin, another flavonoid identified from mung bean, exerts cytotoxic effects on both estrogen receptor-positive and estrogen receptor-negative cells, which coexist in most breast cancers [21]. Breast cancer is the leading cause of cancer-related death among women around the world. Over the last two decades, breast cancer has become the most frequently diagnosed neoplasm in Vietnamese women [29]. A specific cytotoxicity toward MCF-7 cells of NDL could be an interesting perspective to be explored for breast cancer treatment.

Many Vietnamese plants are recognized as possessing anticarcinogenic, antiproliferative and cytotoxic activities. All ingredients of the NDL formula are used for both nutritional and medicinal purposes. A good perspective for investigating this formula was that its ingredients were easily obtainable at low cost, and could be ingested without severe toxicity to normal cells.