In the present study we demonstrated that YXQ-EQ effectively induced apoptosis in SCLC cells, which is in accordance with our previous reports that YXQ-EQ induces apoptosis in pancreatic, prostate and breast cancer cells [4
]. In order to understand the underlying molecular mechanisms of the cytotoxicity of YXQ-EQ on cancer cells, we examined the changes of gene expression profile in SCLC cell line NCI-H82 induced by YXQ-EQ through microarray analysis and identified 39 genes whose expression was altered by YXQ-EQ. Among them, the gene expression levels of oncogenes MYCL1 and DEK were reduced and the levels of apoptotic genes DAPK2 and CIDE-B were increased, which both could inhibit cancer cell proliferation and lead to cell death and play a role in YXQ-EQ-induced cell death/apoptosis. Meanwhile, the expression of cell migration-promoting adhesion molecules CD24 and ITGA9 was found to be downregulated after YXQ-EQ treatment, suggesting YXQ-EQ may have an inhibitory effect on SCLC cell migration. In support of this notion, we have previously reported that YXQ-EQ suppresses the migration and invasion of metastatic breast cancer cell line MDA-MB-231 in vitro [6
]. YXQ-EQ also downregulated the expression of glycolytic enzyme Aldolase A. Thus, our findings indicate that YXQ-EQ may exert anti-lung cancer effect through induction of apoptosis and inhibition of proliferation, migration, and glucose metabolism in cancer cells via simultaneously interfering with the expression of multiple genes involved in various biological pathways and functions.
DAPK2 is a recently identified calcium/calmodulindependent protein kinase that has an intrinsic kinase activity. Increase of intracellular Ca2+
can activate DAPK2 and thereby facilitate apoptosis. Overexpression of DAPK2 induces cell death [34
], and restoration of downregulated DAPK2 expression in Hodgkin lymphoma induces apoptosis [35
]. Aberrant DNA methylation associated with repressing/silencing DAPK2 gene expression was shown in non-small cell lung cancers, nasopharyngeal carcinomas, and colorectal and gastric cancers [36
]. It is not known whether the promoter of DAPK2 gene in SCLC cells is aberrantly methylated to inhibit or silence its expression, but it is reasonable to conjecture that the enhanced expression of DAPK2 in NCI-H82 cells had contributed to the induction of apoptosis in NCI-H82 cells after YXQ-EQ treatment. CIDE-B protein is located in mitochondria and dimerized with other CIDE family members [22
]. It induces apoptosis through cytochrome c release. Like DAPK2, overexpression of CIDE-B induces apoptosis in transfected cells [38
]. Moreover, inactivation of CIDE-B can inhibit CIDE-B-induced apoptosis [38
]. Conceivably, overexpression of CIDE-B in cancer cells would promote cancer cell apoptosis. Elevated expression of DAPK2 and CIDE-B in YXQ-EQ-treated NCI-H82 cells that underwent apoptosis suggests that activating apoptotic pathway(s) which involves DAPK2 and CIDE-B is one of the mechanisms utilized by YXQ-EQ to manifest its cytotoxicity towards cancerous cells.
Besides inducing apoptosis, the other common approach to inhibit cancer cell growth is to downregulate oncogene expression. As transcription factors, Myc family proteins transcriptionally activate several hundred target genes that are involved in diverse biological process [39
]. They increase cell proliferation and cause cell-cycle progression and play a pivotal role in tumorigenesis in numerous human cancers of diverse origin [1
]. Deregulated expressions of c-Myc, N-Myc, and MYCL1 (L-Myc) are evident in many human cancers and shown to promote cancerous cell proliferating [40
]. As a potent oncoprotein that plays a pivotal role as a regulator of tumorigenesis in human cancers, Myc is a very attractive target for cancer treatment. Small-cell lung cancer cell lines, such as NCI-H82, NCI-H209, etc., express MYCL1 [23
]. Specific targeting of MYCL1 by antisense DNA in SCLC cell lines effectively inhibits cell proliferation. The growth inhibition by the antisense DNA is correlated with the level of downregulation of MYCL1 expression [41
]. Retinoic acidinduced growth arrest and apoptosis in NCI-H82 and NCI-H209 cells is associated with substantial reduction of both c-Myc and MYCL1 expression [23
]. It is consistent with the observation that reduction of Myc expression in YXQ-EQ- treated lung cancer cells is correlated with the inhibition of cell proliferation and apoptosis.
Human DEK proto-oncogene encodes a 43-kDa nucleic acid binding phosphoprotein. Though it is ubiquitously expressed in mammalian cell nucleus, the expression level is high in proliferating cells and low in resting and terminally differentiated cells [42
]. It is a senescence inhibitor. Overexpression of DEK protein is implicated in human carcinogenesis [42
]. Since DEK is a senescence inhibitor, reduction of its expression should also help to induce apoptosis in YXQ-EQ-treated cancer cells. It was recently reported that downregulation of DEK expression through RNA interference in human tumor cells result in cell death [44
]. Inhibition of cell proliferation and induction of apoptosis are two interrelated but distinct biological processes. Downregulating the expression of oncogenes MYCL1 and DEK while upregulating the expression of apoptotic DAPK2 and CIDE-B in YXQ-EQ treated cancer cells suggests that YXQ-EQ can inhibit cancer cell growth through suppressing oncogene expression in addition to inducing apoptosis through up-regulation of expression of pro-apoptotic genes.
CD24 is a cell surface molecule used as a B cell lineage differentiation marker. It participates in regulation of both cell proliferation and cell–cell interaction. Overexpression of CD24 in various human cancers facilitates cancer cell metastasis and is strongly associated with a more aggressive course of the disease and poor prognosis. It has been suggested that if the expression of CD24 in cancer cells is downregulated, cancer cell proliferation and metastasis may be inhibited [45
]. It was recently reported that downregulation of CD24 expression in colorectal tumors inhibits tumor growth [46
]. Therefore, CD24 is considered a potential therapeutic target in cancer biology [45
]. ITGA9 is a member of the integrin family. Aberrant upregulation of ITGA9 was found in SCLC [28
], although its precise role in cancer development and progression remains obscure. However, integrins connect cells to components of the extracellular matrix or to counter receptors on other cells mediating cell adhesion, migration, and other cell–cell contact. It is very likely the overexpression of ITGA9 in SCLCs is also linked to the high metastatic nature of lung cancer cells, and ITGA9 has recently been reported to promote tumor cell adhesion and migration [48
]. Thus, CD24 and ITGA9 are very likely among the factors that make the SCLC cells highly prone to metastasis. Downregulation of CD24 and ITGA9 in NCI-H82 cells would affect the capability of cells to migrate, eventually resulting in inhibition of metastasis. Therefore, down-regulation of the expression of CD24 and ITGA9 by YXQ-EQ suggests that YXQ-EQ may inhibit cancer cell migration and metastasis. In agreement, YXQEQ has been shown to repress breast cancer cell migration and invasion [6
Recoverin is one of the paraneoplastic antigens, which is exclusively expressed within photoreceptor cells and retinal bipolar cells under normal circumstances. It is not known why recoverin is aberrantly expressed in tumor cells including lung cancers. Aberrantly expressed recoverin is a highly pathogenic protein [30
]. It is believed that the aberrant expression of recoverin in malignant tumors localized outside the nervous system triggers the production of autoantibodies by the immune system resulting in CAR, an autoimmune syndrome characterized by sudden, progressive loss of vision in association with circulating anti-retinal autoantibodies. Reduction of recoverin in NCIH-82 by YXQ-EQ treatment indicates YXQ-EQ may lower the incidence of SCLC-associated CAR development. Recoverin protein was also found to be associated with caveolin/G-protein-coupled receptor kinases (GRKs) in transformed and cancer cells [49
], which suggest the possible involvement of recoverin in caveolin/GRK-dependent regulation of tumor progression, metastasis, and drug resistance [49
]. YXQ-EQ may affect these processes through downregulation of the expression of recoverin.
Aldolase A, as a glycolytic enzyme, is expressed in developing embryo and in many adult tissues its expression is downregulated [50
]. However, it is overexpressed in human lung cancer and malignant pleural effusion cells [32
]. It has been suggested to be involved in glucose metabolic reprogramming that benefits cancer cells [48
]. Down-regulation of Aldolase A in SCLC cells by YXQ-EQ may interfere with glucose metabolism that is important in cancer cell proliferation and metastasis.
In summary, our data indicate that the expression of genes involved in multiple cellular processes, such as apoptosis, proliferation, migration, adhesion, and glucose/ energy metabolism, is modulated by YXQ-EQ when it induces cell death in cancer cells. These findings provide novel insights into the mechanisms underlying the anticancer effect of YXQ-EQ and important references for further studies.