Development of Cucurbitacin Derivative through In-Silico and In-Vitro Methods to Control Prostate Cancer Cell Proliferation

The present study shows that cucurbitacins are having specific binding affinity for the androgen receptor. This was proved by docking studies where cucurbitacins had a better G score for androgen receptor rather than other target of prostate cancer. Further examination of cucurbitacins for ADME and toxicity properties, we had identified cucurbitacin I was the lead molecule among the cucurbitacins A-U. The androgen receptors are nuclear receptor which is having a great binding affinity for steroidal hormones particularly Dihydrotestosterone. Cucurbitacins are triterpenoids which are the precursor of steroids (David, 1998). This may be the reason for the androgen selectivity. Additionally the presence of numerous keto-, hydroxyl and acetoxy groups are functioning as H-Bond donor/acceptor. These functional groups had formed the essential H-Bond interactions with the androgen receptor. The ligand binding pocket of androgen receptor was made of 12� helix and 2� sheets which folds together to form a hydrophobic ligand binding pocket. The ligandprotein interaction was majorly hydrophobic , however hydrogen bond interaction also plays a major role in specificity. Docking studies and crystallographic studies revealed that ligands which are able to form H-bond interactions with amino acids Asn705, Gln711 and Arg752 andwill have strong affinity towards AR (Karine et al., 2006). Cucurbitacin I forms H-bond interaction at Gln711, Arg 752, Met 749, Asn 705, HOH 108, Trp 741, Met 745, HOH Val 746. Bicalutamide forms H-bond interaction with Gln 711, Arg 752, HOH 108, Asn 705. Recent studies revealed that a full antagonist to AR (ligands which function as an antagonist to wild type and mutated ARs) forms Hbond interactions with Gln711 and Arg752 (Chuangxing et al., 2012). The H-bond interaction with Arg 752, Gln 711 was essential for antagonist activity. The bicalutamide and cucurbitacin I had formed H-bond interaction with those amino acids. The cucurbitacin I and bicalutamide were screened for its cytotoxicity effect in prostate cancer cell lines LNCaP (AR +ve) and PC3 (AR ˗ve). This method of screening in LNCaP and PC3 cells could be used to identify the AR selectivity. Expression of dihyrotestosterone stimulated prostate specific antigen was significantly reduced by cucurbitacin I, this reduction of PSA level (~0.56 fold) was due to antiandrogenic effect of Cucurbitacin I. The PSA is the Prostate cancer marker and AR response element. The cucurbitacin I was found to be 31.4 times selective towards AR positive LNCaP cell line and the standard bicalutamide was 1.48 times selective towards AR positive LNCaP cells. The potency of Cucurbitacin I in LNCaP was found to be 1796 times than bicalutamide. The tremendous potency of cucurbitacin I in LNCaP cells could be due to the synergetic effect of AR antagonism and JAK/STAT inhibition (Alghasham., 2013). Cucurbitacin I is a novel compound that could be further developed to evaluate its in-vivo efficacy. Apoptosis of the Cucurbitacin I treated cells were confirmed by measuring the caspase 3, 8, 9 cleavage activity, Bax/Bcl2 gene expression ratio and Ethidium bromide/Acridine orange staining. Growth factor withdrawal and intracellular stress can induce apoptosis through the intrinsic cell death pathway, while extrinsic apoptosis is initiated through transmembrane death receptors. Initiation and execution of these processes are regulated by the BCL-2 and caspase families of proteins. Activation of the BCL-2 family members Bax and Bak results in mitochondrial outer membrane permeabilization (MOMP) and the release of pro-apoptotic proteins, including cytochrome c, from the inter-membrane space into the cytosol (Matthew et al.,2013). Cytochrome c can then bind apoptotic protease activating factor 1 (Apaf-1) forming the apoptosome and activating caspase-9. Once active, caspase-9 can directly cleave and activate caspase-3. The death receptor-mediated pathway is activated upon ligand binding of cell surface death receptors such as tumor necrosis factor-� (TNF-�), initiating ligandinduced receptor trimerization and the formation of death-inducing signaling complex (DISC). Once caspase-8, the initiator caspase, is recruited in zymogen form to the DISC, it is autocatalytically processed and released from the complex to the cytosol as active tetramer to transactivate a number of downstream executioner caspases including the dominant executioner caspase, caspase-3 (Lee et al. 2003). The significant increase in Caspase 3, 8 and 9 activity of Cucurbitacin I treated cells indicates that apoptosis triggers by both extrinsic and intrinsic pathways. Bcl2 is an anti-apoptotic protein that was found to be increased in Prostate cancer. The anti-apoptotic protein Bcl2, has been associated with the development of androgen-independent prostate cancer due to its high levels of expression in androgen-independent tumors in advanced stages of the pathology (Catz et al., 2003). Reduction of Bcl2 level is essential for apoptosis in prostate cancer cells. The level of Bcl2 mRNA level was significantly reduced in Cucurbitacin I (~0.56 folds) treated LNCaP cells, in comparison with bicalutamide (~0.87 fold) and solvent control (~ 1 fold). The balance between pro- and anti-apoptotic members of this family can determine the cellular fate. Bax and Bcl-2 are the major members of the Bcl-2 family which has potential roles in tumor progression and prognosis of different human malignancies. Bax promotes cell death through permeabilization of mitochondrial outer membrane in response to different cellular stresses. In contrast, Bcl-2 prevents apoptosis by inhibiting the activity of Bax. (Mohan et al., 2012) (Hector et al., 2009). The Bax/Bcl-2 ratio can act as a rheostat which determines cell susceptibility to apoptosis (Raisova et al., 2001). Lower levels of this ratio may lead to resistance of human cancer cells to apoptosis. Thus, the Bax/Bcl-2 ratio can affect tumor progression and aggressiveness. (Hemminki et al., 2010). The Bax/Bcl2 ratio of bicalutamide and Cucurbitacin I was found to be 1.48 and 2.14 respectively, significant increase in Bax/Bcl2 ratio of Cucurbitacin I treated cells in comparison with bicalutamide and solvent control was observed.SUMMARY AND CONCLUSION: The present study identified that the cucurbitacin I had a better binding affinity towards androgen receptor and its H-bond interaction with AR protein was similar as of standard bicalutamide. Cucurbitacin I satisfies the ADME and toxicity parameter studies. Further evaluation of cytotoxity of cucurbitacin I in prostate cancer cell lines LNCaP (AR +ve) and PC3 (AR -ve) reveals that cucurbitacin I is more selective toward AR positive cell line LNCaP than PC3 cells. Moreover the cytotoxic potency of cucurbitacin I in LNCaP cells is 1796 times greater than standard bicalutamide. Reduction of prostate specific antigen in cucurbitacin I treated LNCaP than dihydrotestosterone treated LNCaP cells shows the anti-androgenic property of cucurbitacin I. Apoptosis was confirmed by ethidium bromide/acridine orange staining. Upregulation of caspase 3, 8, 9 in cucurbitacin I treated cells was observed. The mechanism behind the cytotoxicity could be the activation of both external and internal apoptosis pathways. Increased level of Bax/Bcl2 ratio was noted in Cucurbitacin I treated LNCaP cells. The higher levels of this ratio may lead the cancer cells to apoptosis. We conclude that Cucurbitacin I is an effective and potent anti-androgen agent, which cause apoptosis by the activation of external and internal apoptosis pathways and by inhibition of anti-apoptotic protein Bcl2 gene expression. Cucurbitacin I is a novel agent for androgen dependent prostate cancer