DiscussionIn this study, we demonstrated for the first time that combination of HA and APG worked synergistically to reduce cell viability in human malignant neuroblastoma SK-N-DZ, SH-SY5Y, and IMR32 cells. Morphological and biochemical studies indicated that combination of HA and APG caused more apoptosis than either treatment alone in SK-N-DZ cells. Previous studies reported induction of dose-dependent cytotoxicity and apoptosis by HA in leukemia [2] and glioblastoma [19] and by APG in HepG2 [13], prostate cancer 22Rv1 [18], and esophageal squamous cell carcinoma KYSE-510 [12] cells. Combination of APG with gemcitabine in pancreatic cancer [20] and retinoic
Perifosine with genistein in neuroblastoma [21] showed better efficacy than single drug in inhibiting cell viability and inducing apoptosis.The massive accumulation of cells in subG1 phase and Annexin V-FITC/PI binding assay showed the mode of cell death was apoptosis and not necrosis. Earlier reports demonstrated that APG caused G2/M arrest for apoptosis in breast cancer SK-BR-3 cells [15]. Combination of APG with gemcitabine induced apoptosis due to cell cycle arrest in pancreatic cancer cells [20]. A previous study also used Annexin V-FITC/PI assay to report that APG induced apoptosis in prostate carcinoma PC-3 cells [17].We found that combination of HA and APG inhibited expression of VEGF and EGFR. As a potent inducer of angiogenesis, VEGF is associated with cancer cell proliferation, migration, and vascular permeability and it is overexpressed in neuroblastoma [22]. Elevated EGFR is involved in cancer development and progression [23]. VEGF mediated upregulation of Bcl-2 is associated with decreased apoptosis in neuroblastoma cells [24]. APG suppresses EGFR in thyroid cancer cells [25] and inhibits VEGF in prostate carcinoma PC-3 cells [17].Combination therapy worked via extrinsic pathway through Bid cleavage to tBid in SK-N-DZ cells for apoptosis. This finding is correlated with previous reports where APG activated caspase-8 in breast cancer [14] and prostate cancer [26] cells for apoptosis. Caspase-8-dependent cleavage of Bid to tBid provides a link between extrinsic and intrinsic pathways of apoptosis.Levels of expression of Bcl-2 family proteins regulate the commitment of
cells to apoptosis [27]. Levels of expression of the pro-apoptotic Bax and the anti-apoptotic Bcl-2 proteins were altered resulting in significant increase in Bax:Bcl-2 ratio after combination therapy. This observation is in agreement with the earlier studies demonstrating that HA prevents Bcl-2 interaction with Bax to cause apoptosis in glioblastoma cells [19] and APG increases Bax and decreases Bcl-2 expression leading to an increase in Bax:Bcl-2 ratio in prostate [16] and [18] and neuroblastoma [28] cells. Increased Bax:Bcl-2 ratio could trigger the mitochondrial release of pro-apoptotic factors into the cytosol [29] and [30] for apoptosis via intrinsic pathway.The Bcl-2 family proteins regulate the release of cytochrome c from mitochondria [30]. Mitochondrial release of cytochrome c into the cytosol is a pre-condition for activation of caspases for apoptosis through intrinsic pathway [30] and [31]. Western blotting showed that combination therapy caused activation of caspase-3. This finding is correlated with previous studies showing that APG induces apoptosis in human prostate cancer [18] and neuroblastoma [28] cells due to activation of caspase-3 and cleavage of specific substrate [32].The Ca2+-dependent cysteine protease calpain plays an important role in apoptosis [33]. We also found an increase in calpain expression following combination therapy. Co-operation between calpain and caspase-3 has previously been demonstrated in apoptosis [34]. Increased calpain and caspase-3 activities can cause cleavage of 270 kD α-spectrin at specific sites to generate 145 kD SBDP and 120 kD SBDP [33], respectively. We found increases in calpain and caspase-3 activities in SK-N-DZ cells for apoptosis.In conclusion, our results showed that combination of HA and APG worked synergistically in reducing cell viability in malignant neuroblastoma cells and suppressed expression of angiogenic factors and activated both the extrinsic and intrinsic pathways for increasing apoptosis in SK-N-DZ cells.AcknowledgmentsThis work was supported in part by the R01 Grants (NS-57811 and CA-91460) from the National Institutes of Health (Bethesda, MD).
