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Overexpression of drug efflux transporters is commonly associated with multidrug-resistance in cancer therapy. Here for the first time, we investigated the ability of diindolylmethane (DIM), a dietary bioactive rich in cruciferous vegetables, in enhancing the efficacy of Centchroman (CC) by modulating the drug efflux transporters in human breast cancer cells. CC is a selective estrogen receptor modulator, having promising therapeutic efficacy against breast cancer. The combination of DIM and CC synergistically inhibited cell proliferation and induced apoptosis in breast cancer cells. This novel combination has also hindered the stemness of human breast cancer cells. Molecular docking analysis revealed that DIM had shown a strong binding affinity with the substrate-binding sites of ABCB1 (P-gp) and ABCC1 (MRP1) drug-efflux transporters. DIM has increased the intracellular accumulation of Hoechst and Calcein, the substrates of P-gp and MRP1, respectively, in breast cancer cells. Further, DIM stimulates P-gp ATPase activity, which indicates that DIM binds at the substrate-binding domain of P-gp, and thereby inhibits its efflux activity. Intriguingly, DIM enhanced the intracellular concentration of CC by inhibiting the P-gp and MRP1 expression as well as activity. The intracellular retaining of CC has increased its efficacy against breast cancer. Overall, DIM, a dietary bioactive, enhances the anticancer efficiency of CC through modulation of drug efflux ABC-transporters in breast cancer cells. Therefore, DIM-based nutraceuticals and functional foods can be developed as adjuvant therapy against human breast cancer., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest, (Copyright © 2021. Published by Elsevier Inc.)

Aims: Recently, strategies of cancer treatment using combination of agents with distinct molecular mechanism(s) of action are considered more promising due to its high efficacy and reduced systemic toxicity. The study is aimed to improve the efficacy of selective estrogen receptor modulator, Centchroman (CC) by combination with the phytoestrogen Genistein (GN)., Methods: Cytotoxicity was evaluated by Sulforhodamine B assay. Cell cycle analysis was done through flow cytometry. Further, Apoptosis was analyzed using Annexin V/PI staining, tunel assay and electron microscopic examination and verified using western blot analysis. In order to validate the in vitro results, in vivo analysis was performed using 4T1-syngeneic mouse model., Key Findings: In this study, we report that the dietary isoflavone genistein (GN) synergistically improved antineoplasticity of CC in breast cancer by arresting cells at G2/M phase culminating in ROS dependent apoptosis. The combination of CC plus GN caused dysregulation of Bax and Bcl-2 ratio inducing mitochondrial dysfunction, activation of Caspase-3/7, -9 and PARP cleavage. Further, combination significantly suppresses phosphorylation of PI3K/Akt/NF-κB, enhancing apoptosis. Additionally, combination markedly reduced tumor growth compared to CC and GN alone in mouse 4T1 breast tumor model., Significance: Together, these studies suggest that GN represents a potential adjunct molecule whose role in CC induced apoptosis deserves attention., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: Angiogenesis is a recognized hallmark of cancer which promotes cancer cell progression and metastasis. Inhibition of angiogenesis to attenuate cancer growth is becoming desirable strategy for breast cancer management. The present study is aimed to investigate the antiangiogenic efficacy of a novel selective estrogen receptor modulator Centchroman (CC) on human breast cancer cells., Main Methods: Effect of CC on cell viability was evaluated using Sulforhodamine B assay. Endothelial cell proliferation, wound healing, Boyden chamber cell invasion, tube formation and chorioallantoic membrane (CAM) assays were performed to assess the effect of CC on migration, invasion and angiogenesis. Apoptosis, reactive oxygen species generation, caspase-3/7 and intracellular calcium ion level were measured through flow cytometry. Expression levels of HIF-1α, VEGF, VEGFR2, AKT and ERK were assessed by western blot analysis., Key Findings: CC selectively induces apoptosis in human breast cancer cells without affecting non-tumorigenic breast epithelial cells MCF-10A. Moreover, it inhibits migratory, invasive and mammosphere forming potential of breast cancer. Furthermore, CC also inhibited VEGF-induced migration, invasion and tube formation of HUVECs in vitro. CC effectively inhibited neovasculature formation in chicken CAM. Western blot analysis demonstrated that CC inhibited expression of HIF-1α and its downstream target VEGF. Interestingly, CC also suppressed VEGFR2 phosphorylation and consequently attenuated AKT and ERK phosphorylation., Significance: Our findings suggest that CC downregulates VEGF-induced angiogenesis by modulating HIF-1α/VEGFR2 pathway and recommend it (CC) as a potential therapeutic drug for breast cancer treatment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glutaraldehyde and glyoxal cross-linked microspheres were prepared using chitosan with different molecular weights (MWs) and degrees of deacetylation (DDAs) for sustained release of centchroman under physiological conditions. The DDA in chitosan was determined by different methods, and the samples were categorized as chitosan with low (48%), medium (62%), and high (75%) DDA. The size and shape of the microspheres were determined by scanning electron microscopy (SEM), and hydrophobicity was determined by adsorption of Rose Bengal dye on microspheres cross-linked with glutaraldehyde or glyoxal. The effect of MW, DDA, and degree of cross-linking in microspheres was studied on the degree of swelling, as well as by the loading and release of centchroman. The glyoxal cross-linked microspheres were more compact and hydrophobic and showed better sustained release in companion to chitosan microspheres and glutaraldehyde cross-linked microspheres. The linear fractional release of centchroman with the square root of time indicated a Fickian behavior of centchroman, and the microspheres also showed zero-order release kinetics for centchroman.

This study reports assay methodology, tissue distribution, and the basic pharmacokinetic behavior of centchroman and its 7-desmethyl metabolite [7-desmethyl centchroman (DMC)] after a single 12.5 mg/kg po dose in young female rats. Plasma, liver, lung, spleen, uterus, and adipose tissue were collected at various time intervals up to 14 days after dose. Reversed-phase HPLC, coupled with fluorescence detector, was used for simultaneous determination of centchroman and DMC in biosamples. The drug and metabolite were quantitated up to 2 and 5 ng/ml in plasma and 10 and 20 ng/g in tissues, respectively. The assay method was validated in terms of accuracy, precision, interassay, and intraassay variability, and was found to be reliable and reproducible. Peak centchroman levels in all of the tissues were found between 8-12 hr, whereas DMC peaks appeared between 8 and 24 hr, except that in liver the first peak of 1.2 micrograms/g appeared in the 1-hr sample. Tissue-to-plasma concentration ratios of centchroman were > 200 times in the lung; > 100 times in the spleen, liver, and adipose tissue; and > 40 times in the uterus at maxima in each tissue. Similarly, tissue concentrations of DMC were > 350 times in the lung, > 100 times in the liver and spleen, and > 25 times in the uterus and adipose tissue than in the plasma. High tissue-to-plasma concentration ratios of metabolites than the parent drug are indicative of its greater affinity for tissues. Terminal half-life of the centchroman and DMC in plasma were 24.1 and 36.6 hr, respectively. The mean residence time of centchroman was highest in the liver (78.4 hr), followed by the uterus (72.7 hr), adipose tissue (47.5 hr), lung (46 hr), spleen (44.1 hr), and plasma (37.7 hr). The mean residence time of DMC was also highest in the liver (133.7 hr), followed by the uterus (122 hr), adipose tissue (85.2 hr), lung (62.6 hr), spleen (62.6 hr), and plasma (48.2 hr).

We have determined an X-ray crystal structure for the N-methyl iodide derivative of the nonsteroidal contraceptive centchroman. The pendant aromatic substituents on C-3 and C-4 of the chroman system are nearly perpendicular to the plane of the chroman system, an orientation expected in such a chroman, but perturbed to some degree by the gem dimethyl substituents at C-2. Structural superposition with other nonsteroidal antiestrogens, tamoxifen and nafoxidine, shows a similar disposition of the tertiary amine side chains responsible for antagonist activity. The aryl rings also show good superposition, but in contrast to tamoxifen and nafoxidine, which have the potential for ring double bond conjugation, the centchroman aryl rings show a larger dihedral twist. While different superpositions between the enantiomers of centchroman and the bioactive enantiomer of estradiol (d-estradiol, 8 beta,9 alpha,13 beta,14 alpha,17 beta) are possible, when the chroman ring system is positioned over the AB rings of estradiol, then (3R,4R)-centchroman makes the best fit. The aryl substituents in both enantiomers make comparable overlays with the steroidal skeleton, but the axial methyl group at C-2 in (3R,4R)-centchroman is directed downward along the C-7 alpha axis of estradiol, a site where many substituents are known to be well tolerated by the estrogen receptor, while in the 3S,4S-enantiomer, this methyl group is projected upward. Thus, we suggest that the bioactive l-enantiomer of centchroman will have the 3R,4R absolute configuration.

Several compounds structurally related to the triarylethylene antiestrogens, but possessing weak estrogen receptor affinities, were assessed for their ability to interact with the microsomal antiestrogen binding site. While all the compounds tested did interact with this site their relative affinities were somewhat lower than that of tamoxifen. One of these, viz., the cis isomer of centchroman, has however emerged as a selective ligand for the antiestrogen binding site since its estrogen receptor affinity is nearly 50,000 times lower on a relative scale.

Centchroman [dl-3,4-trans-2,2-dimethyl-3-phenyl-4-[p-(beta-pyrrolidinoethoxy)phenyl] - 7-methoxychroman hydrochloride], an antifertility agent under clinical evaluation, has been resolved into its optical enantiomers. The cytosol estrogen receptor binding affinity and estrogenic, antiestrogenic and antiimplantation activities of the two enantiomers have been determined. The enantiomers display a 7-fold difference in receptor affinity, and a corresponding difference in stimulation of the uterine growth and antiimplantation activity was observed in rats.