Caroline Descôteaux

Design, synthesis and biological evaluation of estradiol-chlorambucil hybrids as anticancer agents

A series of estradiol-chlorambucil hybrids was synthesized as anticancer drugs for site-directed chemotherapy of breast cancer. The novel compounds were synthesized in good yields through efficient modifications of estrone at position 16alpha of the steroid nucleus. The newly synthesized compounds were evaluated for their anticancer efficacy in different hormone-dependent and hormone-independent breast cancer cell lines. The novel hybrids showed significant in vitro anticancer activity when compared to chlorambucil. Structure-activity relationship (SAR) reveals the influence of the length of the spacer chain between carrier and drug molecule.

Improved synthesis of unique estradiol-linked platinum(II) complexes showing potent cytocidal activity and affinity for the estrogen receptor alpha and beta

We have recently reported the synthesis of a platinum(II) complex, made of estradiol, the female sex hormone, and a cisplatin analog, an anticancer drug, linked together by an eleven carbon atoms chain. The novel estradiol-Pt(II) hybrid molecule was synthesized in nine chemical steps with 10% overall yield. This new compound has been tested in vitro on estrogen-dependent (MCF-7) and -independent (MDA-MD-231) (ER(+) and ER(-)) cell lines. Interestingly, the biological activity was quite significant, more potent than that of cisplatin, the compound currently used in chemotherapy. The estrogen receptor binding affinity (ERBA) of this compound was very similar to that of 17beta-estradiol (E(2)) on both estrogen receptors (ERs), alpha and beta. In order to further study this type of molecule, we have decided to synthesize several analogs with the same estrogenic scaffold but with various chain lengths separating the estradiol from the toxic part of the molecule. This was planned in order to study the effect of the length of the linking chain on the biological activity of the hybrids. Four E(2)-Pt(II) hybrid molecules having 6-14 carbon atoms linking chain have been synthesized using a new synthetic methodology. They are synthesized in only eight chemical steps with 21% overall yield. The 17beta-estradiol-linked platinum(II) complexes have been tested for their receptor binding affinity as well as for their cytocidal activity on several breast cancer cell lines. The synthesis and biological results are reported herein.

VP-128, a novel oestradiol-platinum(II) hybrid with selective anti-tumour activity towards hormone-dependent breast cancer cells in vivo.

We have previously reported the synthesis of VP-128, a new 17beta-oestradiol (E(2))-linked platinum(II) hybrid with high affinity for oestrogen receptor alpha (ERalpha). In the present study, we have investigated the anti-tumour activity of VP-128 towards breast cancer cells in vitro and in vivo. We used human ERalpha-positive (MCF-7) and -negative (MDA-MB-468) cells as a model for treatment with increasing doses of VP-128, cisplatin or E(2) in vitro and for xenograft experiments in nude mice in vivo. Compared with cisplatin, VP-128 showed markedly improved in vitro and in vivo anti-tumour activity towards ERalpha-positive MCF-7 breast cancer cells, without increased systemic toxicity. In these caspase-3-deficient cells, treatment with VP-128 overcame weak cellular sensitivity to cisplatin in vitro and in vivo. In these cells, only the hybrid induced apoptosis in an ERalpha-dependent manner, inactivated both X-linked inhibitor of apoptosis protein and Akt, and induced selective nuclear accumulation of ERalpha and the expression of ER-regulated genes c-myc and tff1, which was blocked by ERalpha-specific antagonist ICI 282 780. In the case of ERalpha-negative MDA-MB-468 cells, VP-128, but not cisplatin, induced nuclear accumulation of apoptosis-inducing factor and inhibited c-myc expression. However, VP-128 did not show enhanced in vivo anti-tumour activity compared with cisplatin. These results reveal two different modes of action for VP-128 in ERalpha-positive and -negative breast cancer cells, and highlight the promising therapeutic value of this unique E(2)-platinum hybrid for selective targeting of hormone-dependent cancers.

Transfer RNA Bindings to Antitumor Estradiol-Platinum(II) Hybrid and Cisplatin

The anticancer platinum (Pt) drugs exert their antitumor activity by direct or indirect Pt-DNA binding. It has been shown that Pt drugs can induce major DNA damage and minor RNA damage during cancer treatment. A recent report showed that a new anticancer estradiol-Pt(II) hybrid molecule (CD-37) binds DNA bases indirectly, while being more effective than cis-diaminedichloroplatinum(II) (cisplatin) against several types of cancer. In this report, we examine the bindings of CD-37 and cisplatin drugs with transfer RNA (tRNA) in vitro and compare the results to those of the corresponding Pt-DNA complexes. Solutions containing various CD-37 or cisplatin concentrations were reacted with tRNA at physiological pH. Using Fourier transform infrared (FTIR), UV-visible, and circular dichroism spectroscopic methods, the drug binding mode, the binding constant, and RNA structural variations are determined for Pt-tRNA complexes in aqueous solution. Structural analysis showed direct binding of cisplatin drug to guanine and adenine N7 sites, while both direct and indirect interactions of CD-37 with tRNA bases and the backbone phosphate group were observed. The overall binding constants estimated were K(CD-37) = 2.77 (+/-0.90) x 10(4) M(1) and K(cisplatin) = 1.72 (+/-0.50) x 10(4) M(1). Major aggregation of tRNA occurs at high CD-37 concentrations, while RNA remains in the A-family structure.

Synthesis and cytotoxic activity of benzopyran-based platinum(II) complexes.

A series of benzopyran-based platinum complexes of types 4 and 5 were synthesized as potential anticancer agents. The novel compounds were synthesized in several steps using simple and efficient chemistry. The newly synthesized compounds were evaluated for their biological efficacy and showed significant in vitro cytotoxic activity in different hormone-dependent and -independent breast cancer cell lines. Docking and other molecular modeling experiments were also performed for one of the potent compounds, 5f, which showed that both the possible enantiomeric forms (5f with 3R,4R and 5f with 3S,4S) of the molecule have comparable lowest energy (for 5f with 3R,4R, -31.953 kcal/mol and for 5f with 3S,4S, -31.944 kcal/mol). The 3D QSAR was examined for the derivatives of both enantiomeric forms and a novel relationship for the 3S,4S derivatives is discussed.

ERα-targeted therapy in ovarian cancer cells by a novel estradiol-platinum(II) hybrid.

As we previously showed, we have synthesized a new family of 17β-estradiol-platinum(II) hybrids. Earlier studies revealed the VP-128 hybrid to show high efficiency compared with cisplatin toward hormone-dependent breast cancer cells. In the present research, we have studied the antitumor activity of VP-128 in vitro and in vivo against ovarian cancer. In nude mice with ovarian xenografts, VP-128 displayed selective activity toward hormone-dependent tumors and showed higher efficiency than cisplatin to inhibit tumor growth. Similarly, in vitro, transient transfection of estrogen receptor (ER)-α in ERα-negative A2780 cells increased their sensitivity to VP-128-induced apoptosis, confirming the selectivity of VP-128 toward hormone-dependent tumor cells. In agreement, Western blot analysis revealed that VP-128 induced higher caspase-9, caspase-3, and poly (ADP-ribose) polymerase cleavage compared with cisplatin. The activation of caspase-independent apoptosis was also observed in ERα-negative A2780 cells, in which VP-128 rapidly induced the translocation of apoptosis-inducing factor to the nucleus. Conversely, subcellular localization of apoptosis-inducing factor was not modified in ERα-positive Ovcar-3 cells. We also discovered that VP-128 induces autophagy in ovarian cancer cells because of the formation of acidic vesicular organelles (AVOs) and increase of Light Chain 3B-II protein responsible for the formation of autophagosomes; pathways related to autophagy (AKT and mammalian target of rapamycin) were also down-regulated, supporting this mechanism. Finally, the inhibition of autophagy using chloroquine increased VP-128 efficiency, indicating a possible combination therapy. Altogether these results highlight the beneficial value of VP-128 for the treatment of hormone-dependent ovarian cancers and provide preliminary proof of concept for the efficient targeting of ERα- by 17β-estradiol-Pt(II)-linked chemotherapeutic hybrids in these tumors.

Synthesis of D- and L-tyrosine-chlorambucil analogs active against breast cancer cell lines.

A series of D- and L-tyrosine-chlorambucil analogs was synthesized as anticancer drugs for chemotherapy of breast cancer. The novel compounds were synthesized in good yields through efficient modifications of D- and L-tyrosine. The newly synthesized compounds were evaluated for their anticancer efficacy in different hormone-dependent and hormone-independent (ER+ and ER-) breast cancer cell lines. The novel analogs showed significant in vitro anticancer activity when compared to chlorambucil. Structure-activity relationship (SAR) reveals both, the influence of the length of the spacer chain and the stereochemistry of the tyrosine moiety. Interestingly, the D- and L-tyrosinol-chlorambucil derivatives with 10 carbon atoms spacer are selective towards MCF-7 (ER+) breast cancer cell line.

Design of novel tyrosine-nitrogen mustard hybrid molecules active against uterine, ovarian and breast cancer cell lines

L-para-Tyrosine was linked to ortho-hydroxyaniline, meta-hydroxyaniline and para-hydroxyaniline giving three distinct tyrosinamide molecules. The new extended amino acid derivatives were constructed to imitate, in part, the estradiol (E(2), the natural female sex hormone) nucleus. The resulting tyrosinamides were then linked to chlorambucil either directly, or via a 5 and 10 carbon atoms spacer chain. This was done in an attempt to target cancerous cells expressing the estrogen receptor alpha (ERα) and to obtain a more specific chemotherapeutic agent. The tyrosinamide-chlorambucil molecules were designed and synthesized in good yields, according to two different approaches. The novel compounds were evaluated for their anticancer efficacy in hormone-dependent and hormone-independent (ER+; MCF-7 and ER-; MDA-MB-231) breast cancer cell lines. Interestingly, the meta-hydroxyphenyl-tyrosinamide-chlorambucil derivatives were more active than the ortho- and para- analogs. The molecules bearing a 5 carbon atoms spacer were selected for additional biological study using a panel of female cancerous cells; breast (ZR-75-1, MDA-MB-436, MDA-MB-468), ovarian (OVCAR-3, A2780) and uterine (Ishikawa, HEC-1A). It was discovered that for breast cancer cells, the new compounds were up to 4.2 times more active than chlorambucil itself.

SAR study of tyrosine–chlorambucil hybrid regioisomers; synthesis and biological evaluation against breast cancer cell lines

Amino acids were transformed and coupled to chlorambucil, a well-known chemotherapeutic agent, in an attempt to create new anticancer drugs with selectivity for breast cancer cells. Among the amino acids available, tyrosine was selected to act as an estrogenic ligand. It is hypothesized that tyrosine, which shows some structural similitude with estradiol, could possibly mimic the natural hormone and, subsequently, bind to the estrogen receptor. In this exploratory study, several tyrosine-drug conjugates have been designed. Thus, ortho-, meta- and para-tyrosine–chlorambucil analogs were synthesized in order to generate new anticancer drugs with structural diversity, more specifically in regards to the phenol group location. These new analogs were produced in good yield following efficient synthetic methodology. All the tyrosine–chlorambucil hybrids were more effective than the parent drug, chlorambucil. In vitro biological evaluation on estrogen receptor positive and estrogen receptor negative (ER+ and ER−) breast cancer cell lines revealed an enhanced cytotoxic activity for compounds with the phenol function located at position meta. Molecular docking calculations were performed for the pure l-ortho, l-meta- and l-para-tyrosine phenolic regioisomers. The synthesis of all tyrosine–chlorambucil hybrid regioisomers and their biological activity are reported herein. Possible orientations within the targeted protein [estrogen receptor alpha (ERα)] are discussed in relation to the biological activity.

Abstract 4889: ERa-targeted therapy in ovarian cancer cells using novel Estradiol-Platinum(II) hybrids

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Since several cancers in women are estrogen-dependent due to the presence and overexpression of estrogen receptor (ER), we sought to determine the possibility to use ER as a target for directed chemotherapy and have designed unique molecules capable of doing so. We have recently reported the synthesis of a new family of E2-platinum(II) hybrids. Previous study revealed high efficiency of these novel compounds compared to cisplatin towards breast cancer cells which were also selective towards ER+ breast cancer xenografts in nude mice. In the present study, we have investigated, in vitro and in vivo, the antitumor activity of one of the best hybrid VP-128 using ovarian cancer cell (Ovcar-3, Skov-3, A2780 and A2780CP) and investigated its mode of action and its ability to induce apoptosis and autophagy. Annexin V/PI staining using flow cytometry revealed an improved efficiency of VP-128 compared to cisplatin to induce apoptosis of ovarian cancer cells. We also transiently transfected A2780 cells with ERα gene to compare the hybrid in a similar genetic background. The presence of ER in A2780 increased apoptosis induced by VP-128 but not by cisplatin. VP-128 activity was also stronger compared to cisplatin in presence or absence of ER suggesting that the hybrid can act through different mechanisms of action. In vivo, using ovarian cancer cell xenografts in nude mice, we also found that VP-128 had improved antitumour activity compared to cisplatin, and was more specific and selective towards hormone-dependent ER+ than ER- xenografts. To examine VP-128 mechanism of action, we decided to study its ability to induce apoptosis and autophagy. We found that VP-128 induced apoptosis via caspase-dependant (caspase-9; -3 and PARP cleavage) mechanisms but also caspase-independent mechanisms such as AIF translocation to the nucleus, as revealed by Western blot of cytosolic/nuclear cell extracts and immunofluorescence microscopy in A2780(ER-). Conversely, AIF subcellular localization was not modified by VP-128 in Ovcar-3 ER+ cells. Following 8-12h of treatment with VP-128, increased formation of acidic vesicular organelles in cancer cells was observed suggesting the importance of autophagy in this process. We then found by Western blot that LC3B-I was converted to LC3B-II following VP-128 treatment suggesting formation of autophagosome. We then investigated pathways directly (mTor) or indirectly (AKT) possibly involved in the regulation of autophagy. VP-128 decreased the level of both phosphorylated/active mTor and AKT in cancer cells favouring autophagy induction. These results show that VP-128 can not only induce apoptosis but also induce autophagy in ovarian cancer cells. Altogether, this study also highlight the therapeutic value of VP-128 for the treatment of hormone-dependent ovarian cancers, and provide preliminary proof-of-concept for efficient targeting of ERα by E2-Pt(II) linked chemotherapeutic hybrids. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4889. doi:1538-7445.AM2012-4889