Original articleDevelopment of new estradiol-cationic lipid hybrids: Ten-carbon twin chain cationic lipid is a more suitable partner for estradiol to elicit better anticancer activity
Graphical abstract
The present report describes the synthesis and in vitro & in vivo anticancer evaluation of the new cationic lipid-modified estradiol derivatives by varying the carbon chain length between C6 to C14. Our findings disclose that the C10 analogue is a more suitable partner than the existing potent C8 analogue towards imparting selective anticancer activity. IC50 as represented are obtained in B16F10 melanoma cells.
Introduction
Cancer with its resilience evolves several mechanisms for regeneration, survival and proliferation. Hence, combinational approaches that use a blend of therapeutic agents targeting multiple pathways are more effective for treatment than a single agent [1]. There are several clinical examples which illustrate the importance of multiple agent usage in curative cancer therapy [2], [3], [4], [5], [6].
Molecular hybridization technique can produce the effect of combination therapy while using a single agent [7]. Combining two different pharmacophores (i.e., the concept of Molecular hybridization) to combat multifunctional and complex disorders like cancer is an intelligent strategy in medicinal chemistry. Unlike physical combination of parent compounds, this strategy bestows distinct biological activities to the new hybrids [8]. Besides,it confers more efficacy and affinity to the hybrids in comparison to the individual entities.
Cationic lipids are extensively studied non-viral vectors for the delivery of genetic cargoes, including p-DNA, siRNA etc. [9]. The hydrophobic long chain moieties of these amphiphilic molecules are generally one or two fatty acid chains, cholesterol derived moieties etc. Cationic lipids are mainly recognized for their delivery potentials but unlike other small molecules their medicinal properties are under-recognized. Cationic lipids are known to activate several cellular pathways like pro-apoptotic and pro-inflammatory cascades [10], generate reactive oxygen species (ROS) in macrophages or immature B cells [11], [12], activates prolonged calcium [Ca2+]i release from intracellular calcium stores sensitive to thapsigargin [13] etc. Cationic lipids are also known to induce apoptosis, a well programmed biochemical event, in cancer cells [14], [15], [16]. Cationic lipids in conjugation with various pharmacophores such as glycosides, estradiol, haloperidol, benzamide also induce prominent apoptosis through generation of ROS, activation of several kinases (PKC, MAPK), modifications of the mitochondrial membrane potential, release of cytochrome c and caspases, modulation of m-TOR kinase, etc. [17], [18], [19], [20].
From the above discussion it is clear that the pharmacologic potential of the cationic lipids in cancer research is immense. This rationalise, among other possibilities, generation of cationic lipid-based anticancer hybrids. Recently we have reported different classes of cationic lipidated small molecules (Scheme 1a)) that include cationic lipid conjugates of estradiol [18], haloperidol [19] and benzamides [20]. Others have utilized this hybridization concept towards developingemodin-based anticancer agents [21]. From these reports it is obvious that cationic lipid modification of small molecule pharmacophores bestows potent anticancer activity to parent molecules. However, improved biological activity is highly dependent on both small molecule as well as lipid-chain length. Interestingly, not all the combinations of ligands and cationic lipid chain-lengths exhibit anticancer activity. Evidently, there needs to be a specific combination of these two in order to generate potential anticancer agent.
Earlier we have developed cationic lipid estradiol hybrids among widely varying carbon chain lengths. The optimization was done among three molecules, starting from two (ES-C2), eight (ES-C8) and sixteen (ES-C16) carbon chain lengths. Among the three compounds, ES-C8 was found to exhibit potent anti-breast cancer activity [18]. Since ES-C8 is an estradiol-associated molecule we expected that like other estrogen-based drugs (Scheme 1b) this molecule would exhibit usual anti-breast cancer activity against only ER-positive cancers [22], [23], [24]. But the molecule shows activity in breast cancer cells independent of their ER-expression status. We questioned: Does C8 the optimal carbon chain length, which when conjugated with estradiol provide most potent anticancer molecule? The present report describes the synthesis and in vitro & in vivo anticancer evaluation of the newer derivatives of cationic lipid-modified estradiol while varying the carbon chain length between C6 to C14. Our findings disclose that the C10 analogue is a more suitable partner than the existing potent C8 analogue towards imparting selective anticancer activity. The anticancer activity of C10 analogue, i.e., ES-C10 is not only pronounced against ER ± breast cancer cells but also effective against ER + melanoma and pancreatic cancer cells, thereby exhibiting its general utility against cancers of various lineage.
Section snippets
Chemistry
The structures and general synthetic strategies adopted for preparing cationic estradiol conjugates are outlined in Scheme 2. The distinctive novel structural features common to the cationic estradiol derivatives 5a–e disclosed in the present investigation include: (a) The presence of hydrophobic groups which are directly linked to the positively charged nitrogen atom and (b) the presence of ER-binding 17β-estradiol group. The details of the synthetic procedures for the cationic estradiol
Discussion
Estradiol (ES) is the endogenous ligand for ER, which is known to have growth inducing effect on ER + breast cancer cells. As a scientific strategy to develop new class of anticancer drug, we chemically modified ES. This was to be done in such a way so as to modulate the ER function and possibly induce anticancer effect in breast cancer cells. Our data shows that upon chemically conjugating cationic lipid of various chain lengths to ES we indeed obtained potent anticancer effect. In our
Conclusions
In summary, we have identified that cationic lipid with ten carbon chains is the better partner for estradiol in order to elicit superior anticancer activity in different forms of cancer including that of breast, skin and pancreas. Demonstratively, ES-C10 (5c) exhibits potent anticancer activity in melanoma model in vivo. The antitumor effect of 5c is ER-mediated in ER + tumor models. ES-C10, therefore, seems to be an attractive chemotherapeutic substance. We predict that it can possibly be
Chemicals and general procedures for characterizations
Estrone, trypsin, EDTA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), propidium iodide (PI), FITC-labeled annexin V, dimethyl sulfoxide (DMSO), Dulbecco's Modified Eagle Medium (DMEM) and RPMI 1640 were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Lonza, Swiss chemicals. The solvents, salts and common reagents for synthesis were purchased from either Aldrich (Milwaukee, WI, USA) or S. D. Fine Chem (Mumbai, India). All other
Acknowledgments
GS thanks CSIR, Govt. of India, for his doctoral fellowship. RB acknowledges DST, Govt. of India [SR/S1/OC-64/2008]. RB and BSR acknowledge CSIR Network Project ‘ADD’ [CSC 0302] for research support.
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