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The antiproliferative property of TCE was found to be dose dependent with higher doses showing cytotoxic effect but lower doses were seen to induce cell differentiation. The expression of PCNA and cyclin D1 in cells is subject to tight post translational regulation. Various small molecules have been prepared recently to target PCNA and cyclin D1that is found to be over expressed during oncogenesis and tumorigenesis (Donnellan et al 1998, Aggarwal et al 2010, Zongqing et al, 2012, Chandanamali Punchihewa et al 2012). In the current study aqueous ethanolic extract of TCE was observed to downregulate the expressions of PCNA and cyclin D1. As the core molecule of the DNA replisome, PCNA binds to variety of binding partners including S phase specific cyclin-CDKs complex (Wojciech Strzalka et al, 2011, Koundrioukoff et al., 2000) and p21 (Gulbis et al., 1996; Knibiehler et al., 1996). TCE induced inhibition of PCNA and cyclin D1 may suggest that TCE arrests cell cycle at G0/G1 phase as these molecules are important for S- phase execution and G0/G1 phase progression (Bates et al 1994, Hunter et al 1994). This was further confirmed by cell cycle study where growth arrest at G1/S and G2/M phase was detected in TCE treated cells. Higher concentration of TCE arrested the cell phase transition at G0 and G1. Restoring G1 and G2 checkpoints is the key to induce differentiation in neuroblastoma cells as neuronal differentiation is functionally linked to cell cycle regulation which frequently involves arrest of these cell cycle entry points (Georgopoulou et al 2006, Sumrejkanchanakij et al 2006).
Inhibition of neuroblastoma cell proliferation was associated with their differentiation as indicated by enhanced expression of neuron specific cytoskeletal protein markers NF200 and MAP2 as well as cell morphology of TCE treated cells. NF200 and its phosphorylated forms are well established markers for post-mitotic and differentiated neurons (Elder et al. 1998). Other cytoskeletal protein MAP-2 i.e., Microtubule-Associated Protein 2, is also a marker of differentiated neurons and was found to inhibit growth of melanoma cells and induces mitotic defects (Mohammad et al 2005). The differentiation state is accompanied by processes outgrowth which is clearly depicted by α-tubulin staining (Fig. 2A). First detected in the process of identification of neuron specific antibodies the neuronal nuclei antigen i.e., NeuN was found to be exclusively present in post-mitotic differentiating neurons (Kee et al 2009). NeuN has been suggested to be required for the maintenance of the post-mitotic state, or during the process of axonogenesis (Weyer 2003). In TCE treated cells, bands corresponding with classic doublet series of 46-48 kDa along with ≈66 kDa and 70-90 kDa were observed to be significantly (p≤0.05) upregulated. NeuN has been identified as member of RNA binding protein family, Rbfox3 which are highly conserved family of tissue specific splicing regulators and have been shown to regulate a large number of brain and muscle-specific splice choice (Underwood JG et al 2005, Zhou H-L et al 2007). Up-regulated expression of NeuN along with enhanced NF200 and MAP-2 expression clearly indicates induction of differentiation by TCE in IMR32 cells.
Since, mitochondrial dysfunction plays major role in oncogenesis, thus we further studied expression of mitochondrial molecular chaperons HSP70 and mortalin which were found to be significantly elevated in TCE treated cells. HSP70, an ATP-dependent molecular chaperon is involved in translocation (Ryan and Pfanner 2001), folding, assembly, and stabilization of newly synthesized proteins (Bukauet al,2000, Hartl and Hayer-Hartl 2002), refolding of misfolded proteins, degradation of abnormal proteins, and control of the activity of regulatory proteins(Younget al.2003,Neupert and Brunner 2002). Its induction has been reported to be important in the neuronal differentiation and neurite extension (Yang et al 2008). Upregulated expression of senescence marker mortalin, in TCE treated cells was also accompanied by its translocation from perinuclear to pancytoplasmic location and then to nuclear compartment. The pancytoplasmic and nuclear expression of mortalin is indication of differentiating cells undergoing senescence (Wadhwa et al 1993, Deocaris et al 2007, Hsu et al 2008). Recently, a study has demonstrated the correlation between RA induced differentiation and growth arrest and enhanced expression of mortalin along with its binding with Retinoic acid receptors in the process (Shih et al 2012). Thus the upregulation and pancytoplasmic distribution along with nuclear expression of mortalin in TCE treated cells further suggests that TCE treatment may be inducing cellular senescence and mortalin may be an important target molecule for differentiation and senescence inducing signaling cascade. The current data showing increased expression of HSP70 and mortalin with decreased expression of bcl-xl may suggest that TCE induces pro-apoptotic pathway independent of AKT pathway. This idea was supported by TCE retrieval test. When TCE was removed from the media and cells were incubated for another 72 hrs, though control cells continued proliferating and 200 μg/ml TCE treated cells reverted back to proliferation but 300 μg/ml TCE treated cells ceased proliferation and remained differentiated suggesting that higher concentration of TCE induces pro-apoptotic pathway and senescence (Fig. 5B). Expression of HSP70 and mortalin was also highest in 300 μg/ml concentration treated cells, hence suggesting induction of senescence in cells treated with higher dose of TCE.
Most of the neuroblastoma cells, including IMR-32, are resistant to apoptosis and differentiation. Anti-apoptotic protein family bcl2 along with bcl-xl are widely expressed in neuroblastoma cells and have been shown to inhibit chemotherapy induced apoptosis (Hagenbuchner et al 2010, Michael et al 2011, Goldsmith et al 2012). Recent reports on phytochemicals like curcumin, andrographolide, cranberry and proanthocyanidines have established bcl-xl mediated induction of pro-apoptotic pathway on treatment with these natural compounds (Sukumari-Ramesh et al 2011). In our experiments TCE treated cells showed reduction in Bcl-xl expression at both transcriptional and translational levels indicating induction of pro-apoptotic pathway which was further supported by increase in mitochondrial membrane potential as indicated by mitotracker green FM staining of cells and condensation of nuclear proteins in TCE treated cells as indicated by DAPI staining (fig 5 A third panel). Decrease in bcl-xl in TCE treated cells was accompanied by upregualted expression of p-Akt and NFkB, hence showing activation of cell survival pathways in response to TCE treatment to maintain the homeostasis within the cellular environment as the cells undergo irreversible senescence. In addition to its vital role in cell survival, Akt has also been found to regulate neuronal differentiation, and several aspects of neurite outgrowth, including elongation, calibre and branching through PI3K/Akt signaling pathway (Huang et al 2003, Evangelopoulos et al 2005). NFkB is a player with contradictory roles in the cells whose spatial and temporal function at particular time point is dependent upon the type of stimuli of its upstream genes. It plays diverse roles ranging from inhibition of apoptosis, induction of differentiation to induction of cell survival pathway (Feng et al 1999). Expression of NFkB was found to be upregulated in TCE treated cells. Hence it may be suggested that with its differentiation inducing potential, TCE also induces pathways which help in maintaining homeostasis and stabilising cellular machinery.
Another important observation of the current study was that TCE possesses anti-metastatic potential which inhibits invasiveness of malignant cells as indicated by wound scratch assay. In untreated cells, almost 50% of scratched area was repopulated by migrating cells whereas it was only 20% and 10%, respectively, in 200 μg/ml and 300 μg/ml TCE treated cells. This decrease in migration rate of cells also correlated with significant down regulated expression of NCAM and PSA-NCAM in TCE treated cells both at transcriptional and translational levels. NCAM is widely expressed during embryogenesis, down-regulated in the course of differentiation to be re-expressed during progression of some tumors (Seidenfaden et al 2003, Daniel et al 2003). It is highly involved in GDNF mediated signaling which leads to cell migration and axonal outgrowth and play important role during development and injury (Paratcha et al 2003). In most of the tumors, NCAM along with its polysialylated form has been found to be upregulated in tumor cells. Polysialylation of NCAM moiety was found to be decisive for its interaction with its ligands and direct tumor growth by controlling its heterophilic interaction and has role in neo-angiogenesis (Seidenfaden et al 2003, Bussolati et al 2006). PSA-NCAM has been reported to be highly expressed in malignant cells indicating that they have major role inducing metastasis (Tanaka et al 2000, Marie-Claude Amoureux et al 2010, Kataria et al 2011).
As expression of PST has been reported to be governed by cAMP-CREB pathway, which is highly implicated in the regulation of a wide range of biological functions such as growth factor-dependent cell proliferation and survival, glucose homeostasis (Nakagawa et al 2002), and has calcium dependent regulatory mechanism (Bruses et al 1998). Among the various binding partners of NCAM, GAP-43 (neuromodulin/B50) plays key role in directional migration and synaptic plasticity of neurons during development and regeneration (Dent et al 1998, Korshunova et al, 2007). GAP43 has been shown to over express in various peripheral nerve sheath tumors and identified as diagnostic marker for malignant tissues.(Chen et al 2013). Nuclear expression of GAP-43 is typical of undifferentiated cells while in differentiated cells, it is mainly localized in cell processes (Guarnieri et al 2013). In our study majority of cells in TCE treated group showed reduced nuclear expression of GAP-43 while relatively enhanced cytoplasmic expression than untreated cells thus indicating induction of differentiation. Downregulation in expression of MMP-2 and 9 further supported the anti-metastatic potential of TCE as these MMPs have been found induce tumorigenicity (Noujaim et al 2002, Deryugina et al 2006). Reduced rate of cell migration in TCE treated cultures in wound scratch assay may be the collective outcome of differentiation, apoptosis and cell cycle arrest which inhibited their migration as metastatic aggressiveness of the tumor is inversely related to its differentiation status.
Highly recommended in Ayurveda for its general body and mental health promoting activity, scientific literature on Tinospora cordifolia is lacking in the mechanistic aspects of its mode of action and also the identification of its bioactive components. In the light of the present study it may be suggested that TCE acts through multiple cellular pathways for its potential anticancer activity, induces differentiation and inhibits metastatic nature of neuroblastoma cells.