Ardipusilloside I, a natural saponin, has been shown to induce apoptosis in human cervical adenocarcinoma cells, Lewis pulmonary carcinoma, hepatocarcinoma, U87MG cells, and NCI-H460 cells [8, 9, 17]. Our recent data have also demonstrated that ardipusilloside I significantly decreases the viability of human mucoepidermoid carcinoma Mc3 cells in a dose- and time-dependent manner . In order to explore the potential mechanism, the dose 10.0 μg/ml treatment for 48 h which was closed to its IC50 was selected as the main experimental index.
To test whether ardipusilloside I induce apoptosis in human mucoepidermoid carcinoma, cell viability and inhibition were evaluated in Mc3 cells. Morphological studies revealed that Mc3 cells treated with ardipusilloside I displayed obvious apoptotic characteristics such as shrinkage and chromatin margination. Ardipusilloside I induced the apoptosis of Mc3 cells, as detected by annexin V binding and PI dual staining. Apoptosis was confirmed by TEM and nucleosomal DNA ladders, the hallmarks of apoptosis . Our data on Mc3 cells are similar to previous studies in other tumor cell lines in which ardipusilloside I induced tumor cell death with apoptosis [8, 9, 17]. Therefore, ardipusilloside I may be a tumor cell apoptosis inducer.
As ardipusilloside I-induced apoptosis is well documented through analysis of various cancer cells, recent studies investigated the underlying mechanism of its apoptosis action [8, 9, 17]. Xiong et al.  found that ardipusilloside I enhanced expression of Fas and its ligand FasL in glioblastoma cells. Moreover, they indicated that the ardipusilloside I-induced apoptosis in glioblastoma cells depends on the enhanced expression of the FasL/Fas-signaling pathway and is independent of the activation of caspase-8. However, our result showed that Bax protein expression was increased while Bcl-2 expression was decreased after ardipusilloside I treatment. Meanwhile, caspase-3, the downstream caspase, was overexpressed.
Apoptosis, or programmed cell death, is one of the most important processes regulating the balance of cell growth and cell death, and is preferred to necrosis as a mechanism of cancer cell death [19–21]. Resistance to apoptosis is one of the hallmarks of human cancers. The Bcl-2 family of proteins plays an important role in regulating the mitochondrial or intrinsic apoptotic pathway [22, 23]. The apoptotic inhibitory Bcl-2 protein is on the cytoplasmic face of the mitochondrial outer membrane, endoplasmic reticulum, and nuclear envelope, and may participate in mitochondrial permeability transitions . In contrast, Bax is a proapoptotic regulator . Bcl-2 and Bax can be expressed in harmony, and this plays an important role in cell growth and cell death. Cells are active when Bcl-2 is overexpressed while cells die if Bax is overexpressed . It has been reported that overexpression of Bcl-2, which is caused by chromosomal translocation of the Bcl-2 oncogene into the immunoglobulin heavy chain gene locus, is a characteristic feature of human follicular lymphoma . Moreover, the inactivation of the proapoptotic Bax gene resulted by somatic mutations has been identified in certain solid tumors and hematological malignancies. For example, single nucleotide substitutions or frameshift mutations of the Bax gene can occur in mismatch repair-deficient colon cancers or hematopoietic malignancies [28, 29].
In this study, we found that ardipusilloside I could markedly reduce the viability of Mc3 cells. We also found that the proapoptotic regulator protein Bax was overexpressed, and anti-apoptotic Bcl-2 protein expression was reduced following ardipusilloside I treatment. Meanwhile, caspase-3, the downstream caspase, was increased. However, there are still some limitations in this present study. First, because it was hard to make primary cultures and subcultures of normal mucoepidermoid acinar cells, the data of ardipusilloside I on these cells are not included in this study. However, Xiong and colleagues tested the effect of ardipusilloside I on human glioblastoma U87MG cells and normal SVGp12 astrocytes . They found that ardipusilloside I did not affect the viability of SVGp12 astrocytes with a dose of 15 μmol/L, while glioblastoma cells were significantly decreased. Second, caspase-3 and PARP are viewed as key enforcers of apoptosis, and activated caspase-3 can further decompose its critical downstream substrate PARP and ultimately lead to apoptosis [30, 31]. Unfortunately, we cannot determine this from this data, though the upregulation of Bax and caspase-3 protein levels were studied.