Narasimha Swamy

An Estradiol-Porphyrin Conjugate Selectively Localizes Into Estrogen Receptor-Positive Breast Cancer Cells

A conjugate of a C(11)-beta-derivative of estradiol and an asymmetric tetraphenylporphyrin was synthesized to study its potential selective uptake by breast cancer cells naturally over-expressing the nuclear receptor for estrogen (ER). Competitive radioligand binding assays of this conjugate with recombinant ER showed that the conjugate bound to ER in a dose-dependent manner with an EC50 of 274 nM, compared with 1 nM for estradiol, the natural ligand. Cellular uptake studies with ER-positive MCF-7 and ER-negative HS578t human breast cancer cells revealed that, the conjugate was taken up by MCF-7 cells in a dose-dependent manner, which was obliterated by co-incubation with a large excess of estradiol. On the other hand there was very little uptake of the un-conjugated porphyrin by MCF-7 and Hs578t cells. HS578t cells also showed insignificant uptake of the conjugate under the conditions of our experiment. These results strongly suggested that specific interaction between the endogenous ER in MCF-7 cells and the estrogen part of the conjugate enabled these cells to selectively internalize the conjugate over the un-conjugated porphyrin. Therefore, ER-binding conjugates of estradiol and porphyrins could potentially be used for ER-targeted photodynamic therapy of hormone-sensitive cancers of breast, ovary, gonads etc.

Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D3 does not influence this activity

Vitamin D‐binding protein (DBP) is a multi‐functional serum protein that is converted to vitamin D‐binding protein‐macrophage activating factor (DBP‐maf) by post‐translational modification. DBP‐maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone. Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass. Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP‐maf. The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP‐maf treatment. This study evaluates the effects of various forms of DBP‐maf (native, recombinant, and 25‐hydroxyvitamin D3 bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities. Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides. Incubation of osteoclasts with ex vivo generated native DBP‐maf resulted in a dose dependent, statistically significant, activation of the osteoclasts. The activation was similar whether or not the vitamin D binding site of the DBP‐maf was occupied. The level of activity in response to DBP‐maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH)2D3) of osteoclast function. Furthermore, another potent macrophage activating factor, interferon–gamma, had no effect on osteoclast activity. The activated form of a full length recombinant DBP, expressed in E. coli showed no activity in the in vitro assay. Contrary to this finding, baculovirus‐expressed recombinant DBP‐maf demonstrated significant osteoclast activating activity. The normal conversion of DBP to DBP‐maf requires the selective removal of galactose and sialic acid from the third domain of the protein. Hence, the differential effects of the two recombinant forms of DBP‐maf is most likely related to glycosylation; E. coli expressed recombinant DBP is non‐glycosylated, whereas the baculovirus expressed form is glycosylated. These data support the essential role of glycosylation for the osteoclast activating property of DBP‐maf. J. Cell. Biochem. 81:535–546, 2001.

Biochemical and preliminary crystallographic characterization of the vitamin D sterol- and actin-binding by human vitamin D-binding protein.

Vitamin D-binding protein (DBP), a multi-functional serum glycoprotein, has a triple-domain modular structure. Mutation of Trp145 (in Domain I) to Ser decreased 25-OH-D(3)-binding by 80%. Furthermore, recombinant Domain I (1-203) and Domain I + II (1-330) showed specific and strong binding for 25-OH-D(3), but Domain III (375-427) did not, suggesting that only Domains I and II might be required for vitamin D sterol-binding. Past studies have suggested that Domain III is independently capable of binding G-actin. We exploited this apparently independent ligand-binding property of DBP to purify DBP-actin complex from human serum and rabbit muscle actin by 25-OH-D(3) affinity chromatography. Competitive (3)H-25-OH-D(3) binding curves for native DBP and DBP-actin complex were almost identical, further suggesting that vitamin D sterol- and actin-binding activities by DBP might be largely independent of each other. Trypsin treatment of DBP produced a prominent 25 kDa band (Domain I, minus 5 amino acids in N-terminus), while actin was completely fragmented by such treatment. In contrast, tryptic digestion of purified DBP-actin complex showed two prominent bands, 52 (DBP, minus 5 amino acids in the N-terminus) and 34 kDa (actin, starting with amino acid position 69) indicating that DBP, particularly its Domains II and III were protected from trypsin cleavage upon actin-binding. Similarly, actin, except its N-terminus, was also protected from tryptic digestion when complexed with DBP. These results provided the basis for our studies to crystallize DBP-actin complex, which produced a 2.5 A crystal, primitive orthorhombic with unit cell dimensions a=80.2A, b=87.3A, and c=159.6A, P2(1)2(1)2(1) space group, V(m)=2.9. Soaking of crystals of actin-DBP in crystallization buffer containing various concentrations of 25-OH-D(3) resulted in cracking of the crystal, which was probably a reflection of a ligand-induced conformational change in the complex, disrupting crystal contacts. In conclusion, we have provided data to suggest that although binding of 25-OH-D(3) to DBP might result in discrete conformational changes in the holo-protein to influence actin-binding, these binding processes are largely independent of each other in solution.

Synthesis and estrogen receptor binding affinity of a porphyrin-estradiol conjugate for targeted photodynamic therapy of cancer.

A tetraphenylporphyrin-C11-beta-estradiol conjugate has been synthesized. Competitive binding assay of the conjugate with estrogen receptor (ER)-ligand-binding domain showed that the conjugate binds specifically to the protein with high affinity. Potential use of this conjugate to selectively deliver cytotoxic porphyrins to ER-positive cells in various carcinomas is discussed.

Photodynamic cell-kill analysis of breast tumor cells with a tamoxifen-pyropheophorbide conjugate.

We hypothesized that estrogen receptor (ER) in hormone‐sensitive breast cancer cells could be targeted for selective photodynamic killing of tumor cell with antiestrogen‐porphyrin conjugates by combining the over‐expression of ER in hormone‐sensitive breast cancer cells and tumor‐retention property of porphyrin photosensitizers. In this study we describe that a tamoxifen (TAM)‐pyropheophorbide conjugate that specifically binds to ERα, caused selective cell‐kill in MCF‐7 breast cancer cells upon light exposure. Therefore, it is a potential candidate for ER‐targeted photodynamic therapy of cancers (PDT) of tissues and organs that respond to estrogens/antiestrogens. J. Cell. Biochem. 99: 665–670, 2006.

1α,25-Dihydroxyvitamin D3-3β-(2)-bromoacetate, an affinity labeling derivative of 1α,25-dihydroxyvitamin D3 displays strong antiproliferative and cytotoxic behavior in prostate cancer cells

In this report we describe that 1,25(OH)2D3‐3‐BE, a VDR‐affinity labeling analog of 1,25(OH)2D3, showed strong and dose‐dependent growth‐inhibitory effect in several epithelial cells, i.e., keratinocytes (primary cells), MCF‐7 breast cancer, PC‐3, and LNCaP prostate cancer and PZ‐HPV‐7 immortalized normal prostate cell‐lines. Furthermore, 10−6 M of 1,25(OH)2D3‐3‐BE induced apoptosis specifically in LNCaP and PC‐3 cells; and the effect was much less pronounced at lower doses. We also showed that the effect (of 1,25(OH)2D3‐3‐BE) was not due to probable degradation (hydrolysis) of 1,25(OH)2D3‐3‐BE or random interaction of this molecule with cellular proteins. Tissue‐ or cell‐specific action of 1,25(OH)2D3 and its mimics is not common due to the ubiquitous nature of VDR. Furthermore, variable effects of 1,25(OH)2D3 and its analogs in various cell‐lines potentially limits their application as anticancer agents. We showed that 1,25(OH)2D3‐3‐BE displayed similar growth‐inhibitory and cytotoxic activities towards androgen sensitive LNCaP and androgen‐independent PC‐3 cell‐lines. Therefore, these results raise the possibility that 1,25(OH)2D3‐3‐BE or similar VDR‐cross linking analogs of 1,25(OH)2D3 might be considered for further development as potential candidates for prostate cancer. J. Cell. Biochem. 89: 909–916, 2003.

Nuclear estrogen receptor targeted photodynamic therapy : Selective uptake and killing of MCF-7 breast cancer cells by a C17α-alkynylestradiol-porphyrin conjugate

We hypothesized that over‐expression of estrogen receptor (ER) in hormone‐sensitive breast cancer could be harnessed synergistically with the tumor‐migrating effect of porphyrins to selectively deliver estrogen‐porphyrin conjugates into breast tumor cells, and preferentially kill the tumor cells upon exposure to red light. In the present work we synthesized four (4) conjugates of C17‐α‐alkynylestradiol and chlorin e6‐dimethyl ester with varying tether lengths, and showed that all these conjugates specifically bound to recombinant ERα. In a cellular uptake assay with ER‐positive MCF‐7 and ER‐negative MDA‐MB 231 human breast cancer cell‐lines, we observed that one such conjugate (E17‐POR, XIV) was selectively taken up in a dose‐dependent and saturable manner by MCF‐7 cells, but not by MDA‐MB 231 cells. Furthermore, MCF‐7 cells, but not MDA‐MB 231 cells, were selectively and efficiently killed by exposure to red light after incubation with E17‐POR. Therefore, the combination approach, including drug‐ and process modalities has the potential to be applied clinically for hormone‐sensitive cancers in organs where ER is significantly expressed. This could potentially be carried out either as monotherapy involving a photo‐induced selective destruction of tumor cells and/or adjuvant therapy in post‐surgical treatment for the destruction of residual cancer cells in tissues surrounding the tumor. J. Cell. Biochem. 99: 966–977, 2006.

Development of an affinity-driven cross-linker : Isolation of a vitamin D receptor associated factor

A vitamin D analogue containing an affinity and a photoaffinity probe (affinity-driven cross-linker, Double Label) was synthesized. An unknown factor, associated with vitamin D receptor (VDR), was isolated from rat liver nuclear extract using a GST-VDR-ligand-binding domain fusion protein (GST-VDR-LBD), affinity labeled with Double Label, and protein-protein cross-linking by photolysis.

C-6 functionalized analogs of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3: synthesis and binding analysis with vitamin D-binding protein and vitamin D receptor

In this article, we describe the development of a general synthetic strategy to functionalize the C-6 position of vitamin D3 and its biologically important metabolites, i.e. 25-hydroxyvitamin D3 (25-OH-D3) and 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We employed Mazurs cyclovitamin D method to synthesize vitamin D3 analogs with several functionalities at the C-6 position. In addition, we synthesized 6-(3-hydroxypropyl) and 6-[(2-bromoacetoxy)propyl] derivatives of 25-OH-D3 15 and 16, respectively, and 6-(3-hydroxypropyl) derivative of 1,25(OH)2D3 17. Competitive binding assays of 15-17 with human serum vitamin D-binding protein showed that all these analogs specifically bound to this protein, although with significantly lower affinity than the 25-OH-D3, the strongest natural binder, but with comparable affinity with 1,25(OH)2D3, the hormone. On the other hand, 6-[3-hydroxypropyl], 1alpha,25-dihydroxyvitamin D3 17 did not show any specific binding for recombinant nuclear vitamin D receptor. These results indicated that the region containing the C-6 position of the parent seco-steroid [1,25(OH)2D3] may be an important recognition marker towards vitamin D receptor binding. Information, delineated in this article, will be important for evaluating structure-activity relationship in synthetic analogs of vitamin D and its metabolites.

Why do we need a three-dimensional architecture of the ligand-binding domain of the nuclear 1α, 25-dihydroxyvitamin D3 receptor?

Abstract Highly specific binding of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) by vitamin D receptor (VDR), a nuclear transcriptional factor, activates a genomic mechanism that is manifested in the multiple biologic properties of 1α,25(OH)2D3. Numerous synthetic analogs of 1α,25(OH)2D3 have been employed to study the interaction between 1α,25(OH)2D3 and VDR, and to identify structural markers in 1α,25(OH)2D3 that are important for VDR-binding. On the other hand the three-dimensional structure of VDR remained elusive till very recently. In the present study we employed affinity labeling (by 1α,25-dihydroxyvitamin D3-3-bromoacetate, 1α,25(OH)2D3-3-BE) of VDR to identify C288 as the anchoring residue for the 3-hydroxyl group of 1α,25(OH)2D3 inside the ligand-binding domain of VDR (VDR-LBD). In addition we carried out mutation/hormone-binding analyses to determine the importance of M284 and W286 toward hormone binding. We incorporated this information with the three-dimensional structure of the LBD of progesterone receptor to develop a homology-extension model of VDR-LBD. This model identified several amino acid residues as ligand-contact points inside the LBD. Mutational and hormone-binding analyses of these residues verified the structure-functional authenticity of this model, in comparison with the crystal structure of VDR, bound to 1α,25(OH)2D3.