Sandra Kuntz

Synthesis of new troglitazone derivatives: Anti-proliferative activity in breast cancer cell lines and preliminary toxicological study

Breast cancer is the most prevalent cancer in women. The development of resistances to therapeutic agents and the absence of targeted therapy for triple negative breast cancer motivate the search for alternative treatments. With this aim in mind, we synthesised new derivatives of troglitazone, a compound which was formerly used as an anti-diabetic agent and which exhibits anti-proliferative activity on various cancer cell lines. Among the compounds prepared, some displayed micromolar activity against hormone-dependent and hormone-independent breast cancer cells. Furthermore, the influence of the compounds on the viability of primary cultures of human hepatocytes was evaluated. This enabled us to obtain for the first time interesting structure-toxicity relationships in this family of compounds, resulting in 6b and 8b, which show good anti-proliferative activities and poor toxicity towards hepatocytes, compared to troglitazone.

New troglitazone derivatives devoid of PPARγ agonist activity display an increased antiproliferative effect in both hormone-dependent and hormone-independent breast cancer cell lines.

Numerous recent studies indicate that most anticancer effects of PPARγ agonists like thiazolidinediones are the result of PPARγ-independent pathways. These conclusions were obtained by several approaches including the use of thiazolidinedione derivatives like Δ2-Troglitazone (Δ2-TGZ) that does not activate PPARγ. Since biotinylation has been proposed as a mechanism able to increase the specificity of drug delivery to cancer cells which could express a high level of vitamin receptor, a biotinylated derivative of Δ2-TGZ (bΔ2-TGZ) has been synthetized. In the present article, we have studied the in vitro effects of this molecule on both hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231) breast cancer cells. In both cell lines, bΔ2-TGZ was more efficient than Δ2-TGZ to decrease cell viability. bΔ2-TGZ was also more potent than Δ2-TGZ to induce the proteasomal degradation of cyclin D1 in both cell lines and those of ERα in MCF-7 cells. However, in competition experiments, the presence of free biotin in the culture medium did not decrease the antiproliferative action of bΔ2-TGZ. Besides, other compounds that had no biotin but that were substituted at the same position of the phenolic group of the chromane moiety of Δ2-TGZ decreased cell viability similarly to bΔ2-TGZ. Hence, we concluded that the increased antiproliferative action of bΔ2-TGZ was not due to biotin itself but to the functionalization of the terminal hydroxyl group. This should be taken into account for the design of new thiazolidinedione derivatives able to affect not only hormone-dependent but also hormone-independent breast cancer cells in a PPARγ-independent pathway.

Sexual Development of the Urodele Amphibian Pleurodeles waltl

Pleurodeles waltl is a urodele amphibian that displays a ZZ/ZW genetic mode of sex determination involving a putative W-borne dominant determinant. This determining pathway can be environmentally inhibited since heat treated ZW larvae undergo a functional female to male sex reversal. Moreover, both genetic sexes can be reversed by treatment of larvae with steroid hormones suggesting they are the major players in the differentiation process. Indeed we demonstrated that i) aromatase expression and activity increase just before ovarian differentiation, ii) aromatase inhibitors induce a female to male sex reversal, iii) estrogens induce male to female sex reversal whereas the opposite is obtained with non-aromatizable androgens, iv) steroidogenic factor 1 and estrogen receptor alpha both display a female-enriched expression following the increase in aromatase activity. The role of endogenous hormones was investigated in a parabiosis model. Surprisingly, in ZW/ZZ associations, the ZW gonad could not differentiate suggesting that the ZZ parabiont produces an inhibiting factor, prior to ovarian differentiation. The role of AMH in this process is discussed, keeping in mind that Müllerian ducts are maintained in males. The development of antibodies and new molecular tools in the near future should help us to better understand the sexual development of this vertebrate.

Temporal and spatial SOX9 expression patterns in the course of gonad development of the caudate amphibian Pleurodeles waltl

The SOX family of transcription factors is thought to regulate gene expression in a wide variety of developmental processes. Namely, SOX9 expression is conserved in vertebrate sex determination or differentiation. Nevertheless, information about caudate amphibians is lacking. In this study, we provide data on Pleurodeles waltl, a species that displays a ZZ/ZW genetic mode of sex determination and a temperature-dependent mechanism of female-to-male sex reversal. Phylogenetic analysis of SOX9 P. waltl ortholog reveals that the deduced protein segregates from the group of anuran and could be more closely related to amniote than to anamniote. However, SOX9 lacks the PQA-rich domain present in amniotes. In larvae, SOX9 is expressed in both sexes in gonad-mesonephros complexes as soon as stage 42, before gonad differentiation. At stage 54(60d) at which testis differentiation is already in progress, analyses of isolated gonads reveal a male-enriched expression of SOX9, which was quantified by real-time PCR. At the end of metamorphosis (stage 56), SOX9 shows a nuclear localization only in the testis. In adults, SOX9 is still expressed in testes and ovaries. In the ovary, SOX9 is found in oocytes from stage I to stage VI but it is never detected in the nucleus. Our results suggest that in P. waltl, like in non mammalian vertebrates, SOX9 could play a role during the late phase of gonad differentiation rather than in sex determination. Its role in germ cells of the adult ovary has still to be elucidated.

PPARγ-inactive Δ2-troglitazone independently triggers ER stress and apoptosis in breast cancer cells

Our aim was to better understand peroxisome proliferator‐activated receptor gamma (PPARγ)‐independent pathways involved in anti‐cancer effects of thiazolidinediones (TZDs). We focused on Δ2‐troglitazone (Δ2‐TGZ), a PPARγ inactive TZD that affects breast cancer cell viability. Appearance of TUNEL positive cells, changes in mitochondrial membrane potential, cleavage of poly(ADP‐ribose) polymerase (PARP)‐1 and caspase‐7 revealed that apoptosis occurred in both hormone‐dependent MCF7 and hormone‐independent MDA‐MB‐231 breast cancer cells after 24 and 48 h of treatment. A microarray study identified endoplasmic reticulum (ER) stress as an essential cellular function since many genes involved in ER stress were upregulated in MCF7 cells following Δ2‐TGZ treatment. Δ2‐TGZ‐induced ER stress was further confirmed in MCF7 cells by phosphorylation of pancreatic endoplasmic reticulum kinase‐like endoplasmic reticulum kinase (PERK) and its target eIF2α after 1.5 h, rapid increase in activating transcription factor (ATF) 3 mRNA levels, splicing of X‐box binding protein 1 (XBP1) after 3 h, accumulation of binding immunogloblulin protein (BiP) and CCAAT‐enhancer‐binding protein homologous protein (CHOP) after 6 h. Immunofluorescence microscopy indicated that CHOP was relocalized to the nucleus of treated cells. Similarly, in MDA‐MB‐231 cells, overexpression of ATF3, splicing of XBP1, and accumulation of BiP and CHOP were observed following Δ2‐TGZ treatment. In MCF7 cells, knock‐down of CHOP or the inhibition of c‐Jun N‐terminal kinase (JNK) did not impair cleavage of PARP‐1 and caspase‐7. Altogether, our results show that ER stress is an early response of major types of breast cancer cells to Δ2‐TGZ, prior to, but not causative of apoptosis.

Energy restriction mimetic agents to target cancer cells: comparison between 2-deoxyglucose and thiazolidinediones.

The use of energy restriction mimetic agents (ERMAs) to selectively target cancer cells addicted to glycolysis could be a promising therapeutic approach. Thiazolidinediones (TZDs) are synthetic agonists of the nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ that were developed to treat type II diabetes. These compounds also display anticancer effects which appear mainly to be independent of their PPARγ agonist activity but the molecular mechanisms involved in the anticancer action are not yet well understood. Results obtained on ciglitazone derivatives, mainly in prostate cancer cell models, suggest that these compounds could act as ERMAs. In the present paper, we introduce how compounds like 2-deoxyglucose target the Warburg effect and then we discuss the possibility that the PPARγ-independent effects of various TZD could result from their action as ERMAs.

PPARγ-independent activity of thiazolidinediones: a promising mechanism of action for new anticancer drugs?

Thiazolidinediones are agonists of the nuclear receptor Peroxisome Proliferator Activated Receptor gamma (PPARγ). These synthetic compounds are known for a long time to have anticancer potencies and numerous studies have been performed to understand their mechanism of action. An increasing amount of converging results indicate that most of these mechanisms appear in a PPARγ-independent manner. This was suggested by the observation of effects in cell types devoid of PPARγ and by the absence of correlation between agonist efficiencies and the intensity of the effects. The PPARγ-independence was demonstrated by various experimental approaches using PPARγ antagonists, RNA interference targeting PPARγ or thiazolidinedione derivatives devoid of PPARγ agonist activity. Here, we review the studies that describe PPARγ-independent changes including ionic changes (intracellular pH and Ca2+), reactive oxygen species production, Mitogen Activated Protein Kinases activation, endoplasmic reticulum stress and proteasomal degradation of key proteins. The relationships between these events that occur either early or late after thiazolidinedione exposure and their possible involvement in the antineoplastic effect are discussed. We conclude about the possibility to take advantage from these data to develop new thiazolidinedione derivatives usable as anticancer drugs.

Synthesis and Anti-Proliferative Activity of New Biphenyle-Benzylidenethiazolidine- 2,4-dione Bis-Adducts Containing Various Heterocyclic Cores

In the course of our ongoing program dedicated to the synthesis of anti-proliferative compounds, we prepared new troglitazone derivatives bearing a biphenyle moiety. The chromane heterocycle was further replaced by imidazole and triazole derivatives. Many compounds exhibited fair to high activity towards various cancer cell lines. Among them, compound 17b reduced cell viability leading to only 22-34% viable cells in four cancer cell lines at 10 mu M, but unfortunately also led to a low (13%) cell viability of non-malignant primary cultured human hepatocytes at 200 mu M. Interestingly, compound 11b also reduced cell viability in colon and liver cancer cell lines (29% and 24% cell viability respectively at 10 mu M), but maintained a high cell viability of non-malignant hepatocytes (reaching 71% cell viability at 200 mu M), thus exhibiting a huge selectivity.

Reprofiling of Troglitazone Towards More Active and Less Toxic Derivatives: A New Hope for Cancer Treatment?

The existence of unresponsive tumors and the appearance of resistant tumors during the course of treatments both justify that we increase urgently the panel of pharmacological molecules able to fight cancer. An interesting strategy is drug reprofiling (also known as drug repositioning, drug repurposing or drug retasking) that consists of identifying and developing new uses for existing drugs. This review illustrates drug reprofiling with troglitazone (TGZ), a synthetic PPARγ agonist initially used for the treatment of type II diabetes. The fact that TGZ also displays anticancer effects is known since the end of the nineties but its development as an anticancer agent was slowed down due to hepatotoxic side effects. Part of the knowledge available for TGZ, mainly the molecular basis for PPARγ activation, its metabolization pathways and the side effects on hepatocytes, were taken into account to elaborate new molecules. Key findings were that unsaturated TGZ derivatives, when compared to TGZ, do not activate PPARγ, exhibit a higher efficiency on cancer cells and a lower toxicity towards hepatocytes. However, a weakness is that the mechanisms involved in the anticancer effects are still not completely understood and that the efficiency of such derivatives has not yet been completely studied in vivo. Data about this point should become available very soon from animal models and this will be a prerequisite to initiate clinical trials with these potential new anticancer drugs developed from a drug repurposing strategy.

246: Effects of the potential energy restriction mimetic agent delta2-troglitazone in breast cancer cells

Background: The development of de novo and acquired resistance to anticancer therapies and the absence of targeted therapy for some types of tumors are strong motivations for discovering new therapeutic agents. Thiazolidinediones (TZD) are studied in this context although they were initially designed for the treatment of type II diabetes due to their PPAR gamma agonist activity. They display anticancer effects that are independent of PPAR gamma, but that are not well understood. In prostate cancer cells, TZD derivatives were shown to act as energy restriction mimetic agents. Our team studies the effects of delta2 troglitazone (delta2-TGZ), a TZD devoid of PPAR gamma agonist activity, on breast cancer cell lines. The aim of this work was to better characterize delta2-TGZ-induced cell death and to understand the underlying molecular events.Material and Methods: Delta2-TGZ was obtained by chemical synthesis. We used hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231) breast cancer cell lines. Gene expression was studied by RT-PCR, western blotting and immunocytochemistry. RNA interference was used for gene silencing.Results and discussion: Delta2-TGZ-induced endoplasmic reticulum (ER) stress in MCF-7 cells as shown by phosphorylation of Pancreatic endoplasmic reticulum kinase-like Endoplasmic Reticulum Kinase (PERK) detected after 1.5 hours, splicing of XBP1 (X-box binding protein 1) after 3 hours, accumulation of the chaperone BiP (Binding immunogloblulin protein) and the pro-apoptotic protein CHOP (CCAAT-enhancer-binding protein homologous protein) after 6 hours. CHOP was located in the nucleus of treated cells. Similar events were observed in MDA-MB-231 cells exposed to delta2-TGZ. In both cell lines cleavage of PARP-1 and caspase-7 revealed apoptosis. In MCF-7 cells, knock-down of CHOP or inhibition of c-Jun N-terminal kinase (JNK) did not impair apoptosis. Preliminary results also indicated that AMP-dependent kinase (AMPK) is activated early in MCF-7 cells after delta2-TGZ treatment. Conclusions: This work contributes to a better understanding of the PPARgamma-independent effects of TZD in breast cancer cells. ER stress is an early response to delta2-TGZ, occurring prior to, but not causative of apoptosis. We have now to confirm whether AMPK phosphorylation is a marker of the energy restriction mimetic action of delta2-TGZ and if this is responsible for ER stress and apoptosis.