Somatostatin subtype-2 receptor-targeted metal-based anticancer complexes

Conjugates of a dicarba analogue of octreotide, a potent somatostatin agonist whose receptors are overexpressed on tumor cells, with [PtCl 2(dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (3), [(η 6-bip)Os(4-CO 2-pico)Cl] (bip = biphenyl, pico = picolinate) (4), [(η 6-p-cym)RuCl(dap)] + (p-cym = p-cymene) (5), and [(η 6-p-cym)RuCl(imidazole-CO 2H)(PPh 3)] + (6), were synthesized by using a solid-phase approach. Conjugates 3-5 readily underwent hydrolysis and DNA binding, whereas conjugate 6 was inert to ligand substitution. NMR spectroscopy and molecular dynamics calculations showed that conjugate formation does not perturb the overall peptide structure. Only 6 exhibited antiproliferative activity in human tumor cells (IC 50 = 63 ± 2 μ in MCF-7 cells and IC 50 = 26 ± 3 μ in DU-145 cells) with active participation of somatostatin receptors in cellular uptake. Similar cytotoxic activity was found in a normal cell line (IC 50 = 45 ± 2.6 μ in CHO cells), which can be attributed to a similar level of expression of somatostatin subtype-2 receptor. These studies provide new insights into the effect of receptor-binding peptide conjugation on the activity of metal-based anticancer drugs, and demonstrate the potential of such hybrid compounds to target tumor cells specifically. © 2012 American Chemical Society

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides

Anàlisi de la vehiculització de compostos metàl·lics com a agents antitumorals i de la família HER-EGF en la resistència als tractaments oncològics

Most current chemotherapeutic agents are of limited use due to their side effects and the resistance to treatment that tumour cells develop. In this thesis, we have contributed to the knowledge of these limitations through two studies. First, we have demonstrated that the union of platinum, ruthenium and osmium complexes to octreotide and folic acid analogues has great potential to direct these conjugates specifically to the tumour cells. Second, we have established that one of the resistance mechanisms involved in the low response of the HER-EGF agents in prostate cancer is based on the alternative HER-EGF receptor and ligand overexpression. Specifically, we have demonstrated that the overexpression and autocrine activation of HER3 play a key role in that processMolts dels agents utilitzats en la quimioteràpia actual tenen un ús limitat com a conseqüència dels efectes adversos que produeixen i de les resistències que desenvolupen les cèl·lules tumorals a aquests tractaments. En aquesta tesi, s’ha contribuït en el coneixement d’aquestes limitacions a través de dos estudis. D’una banda, s’ha demostrat que la unió de complexos metàl·lics de platí, ruteni i osmi a anàlegs de l’octreotide i l’àcid fòlic té un elevat potencial per dirigir aquests conjugats específicament cap les cèl·lules tumorals. D’altra banda, s’ha provat que un dels mecanismes de resistència implicats en la baixa resposta dels agents dirigits a inhibir la via HER-EGF en el càncer de pròstata es basa en la sobreexpressió de receptors i lligands de la pròpia via alternatius a l’inhibit; en concret, s’ha demostrat que la sobreexpressió i l’activació autocrina del receptor HER3 tenen un paper clau en aquest procé

Androgen-independent prostate cancer cells circumvent EGFR inhibition by overexpression of alternative HER receptors and ligands

The deregulation of the epidermal growth factor receptor (EGFR) pathway plays a major role in the pathogenesis of prostate cancer (PCa). However, therapies targeting EGFR have demonstrated limited effectiveness in PCa. A potential mechanism to overcome EGFR blockade in cancer cells is the autocrine activation of alternative receptors of the human EGFR (HER) family through the overexpression of the HER receptors and ligands. In the present study, we were interested in analyzing if this intrinsic resistance mechanism might contribute to the inefficacy of EGFR inhibitors in PCa. To this end, we selected two androgen-independent human prostate carcinoma cell lines (DU145 and PC3) and established DU145 erlotinib-resistant cells (DUErR). Cells were treated with three EGFR inhibitors (cetuximab, gefinitib and erlotinib) and the sensitivity to each treatment was assessed. The gene expression of the four EGFR/HER receptors and seven ligands of the HER family was analyzed by real-time PCR prior to and after each treatment. The receptors expression and activation were further characterized by flow cytometry and western blot analysis. EGFR inhibition rapidly induced enhanced gene expression of the EGF, betacellulin and neuregulin-1 ligands along with HER2, HER3 and HER4 receptors in the DU145 cells. In contrast, slight changes were observed in the PC3 cells, which are defective in the phosphatase and tensin homolog (PTEN) tumor suppressor gene. In the erlotinib-resistant DUErR cells, the expression of HER2 and HER3 was increased at mRNA and protein levels together with neuregulin-1, leading to enhanced HER3 phosphorylation and the activation of the downstream PI3K/Akt survival pathway. HER3 blockage by a monoclonal antibody restored the cytostatic activity of erlotinib in DUErR cells. Our results confirm that the overexpression and autocrine activation of HER3 play a key role in mediating the resistance to EGFR inhibitors in androgen-independent PCa cell

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η⁶-<i>p</i>-cym)­RuCl­(PPh₃)₂]⁺, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with <i>N</i>,<i>N</i>′-di-Boc-<i>N</i>″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin–ruthenium conjugate (<b>2</b>) displayed moderate antiproliferative activity in both cancer cell lines (IC₅₀ ≈ 80 μM), whereas the neamine conjugate (<b>4</b>) was inactive (IC₅₀ ≈ 200 μM). However, the guanidinylated analogue of the neomycin–ruthenium conjugate (<b>3</b>) required much lower concentrations than the parent conjugate for equal effect (IC₅₀ = 7.17 μM in DU-145 and IC₅₀ = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (<b>3</b> ≫ <b>2</b> > <b>4</b>), IC₅₀ values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC₅₀ = 49.4 μM for <b>2</b>) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC₅₀ = 12.75 μM for <b>3</b>) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates <b>2</b> and <b>3</b> revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of <b>3</b>. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin–ruthenium conjugate (<b>3</b>) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin–ruthenium conjugate (<b>2</b>). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium­(II) arene derivatives to guanidinoglycosides

Dual Fatty Acid Synthase and HER2 Signaling Blockade Shows Marked Antitumor Activity against Breast Cancer Models Resistant to Anti-HER2 Drugs

Blocking the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of HER2-positive breast carcinoma cells. The hypothesis is that blocking FASN, in combination with anti- HER2 signaling agents, would be an effective antitumor strategy in preclinical HER2+ breast cancer models of trastuzumab and lapatinib resistance. We developed and molecularly characterized in vitro HER2+ models of resistance to trastuzumab (SKTR), lapatinib (SKLR) and both (SKLTR). The cellular interactions of combining anti-FASN polyphenolic compounds (EGCG and the synthetic G28UCM) with anti-HER2 signaling drugs (trastuzumab plus pertuzumab and temsirolimus) were analyzed. Tumor growth inhibition after treatment with EGCG, pertuzumab, temsirolimus or the combination was evaluated in two in vivo orthoxenopatients: one derived from a HER2+ patient and another from a patient who relapsed on trastuzumab and lapatinib-based therapy. SKTR, SKLR and SKLTR showed hyperactivation of EGFR and p-ERK1/2 and PI3KCA mutations. Dual-resistant cells (SKLTR) also showed hyperactivation of HER4 and recovered levels of p-AKT compared with mono-resistant cells. mTOR, p-mTOR and FASN expression remained stable in SKTR, SKLR and SKLTR. In vitro, anti-FASN compounds plus pertuzumab showed synergistic interactions in lapatinib- and dual- resistant cells and improved the results of pertuzumab plus trastuzumab co-treatment. FASN inhibitors combined with temsirolimus displayed the strongest synergistic interactions in resistant cells. In vivo, both orthoxenopatients showed strong response to the antitumor activity of the combination of EGCG with pertuzumab or temsirolimus, without signs of toxicity. We showed that the simultaneous blockad

Somatostatin Subtype‑2 Receptor-Targeted Metal-Based Anticancer Complexes

Conjugates of a dicarba analogue of octreotide, a potent somatostatin agonist whose receptors are overexpressed on tumor cells, with [PtCl₂(dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (<b>3</b>), [(η⁶-bip)­Os­(4-CO₂-pico)­Cl] (bip = biphenyl, pico = picolinate) (<b>4</b>), [(η⁶-<i>p</i>-cym)­RuCl­(dap)]⁺ (<i>p</i>-cym = <i>p</i>-cymene) (<b>5</b>), and [(η⁶-<i>p</i>-cym)­RuCl­(imidazole-CO₂H)­(PPh₃)]⁺ (<b>6</b>), were synthesized by using a solid-phase approach. Conjugates <b>3</b>–<b>5</b> readily underwent hydrolysis and DNA binding, whereas conjugate <b>6</b> was inert to ligand substitution. NMR spectroscopy and molecular dynamics calculations showed that conjugate formation does not perturb the overall peptide structure. Only <b>6</b> exhibited antiproliferative activity in human tumor cells (IC₅₀ = 63 ± 2 μM in MCF-7 cells and IC₅₀ = 26 ± 3 μM in DU-145 cells) with active participation of somatostatin receptors in cellular uptake. Similar cytotoxic activity was found in a normal cell line (IC₅₀ = 45 ± 2.6 μM in CHO cells), which can be attributed to a similar level of expression of somatostatin subtype-2 receptor. These studies provide new insights into the effect of receptor-binding peptide conjugation on the activity of metal-based anticancer drugs, and demonstrate the potential of such hybrid compounds to target tumor cells specifically

Dual Fatty Acid Synthase and HER2 Signaling Blockade Shows Marked Antitumor Activity against Breast Cancer Models Resistant to Anti-HER2 Drugs

Blocking the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of HER2-positive breast carcinoma cells. The hypothesis is that blocking FASN, in combination with anti-HER2 signaling agents, would be an effective antitumor strategy in preclinical HER2+ breast cancer models of trastuzumab and lapatinib resistance. We developed and molecularly characterized in vitro HER2+ models of resistance to trastuzumab (SKTR), lapatinib (SKLR) and both (SKLTR). The cellular interactions of combining anti-FASN polyphenolic compounds (EGCG and the synthetic G28UCM) with anti-HER2 signaling drugs (trastuzumab plus pertuzumab and temsirolimus) were analyzed. Tumor growth inhibition after treatment with EGCG, pertuzumab, temsirolimus or the combination was evaluated in two in vivo orthoxenopatients: one derived from a HER2+ patient and another from a patient who relapsed on trastuzumab and lapatinib-based therapy. SKTR, SKLR and SKLTR showed hyperactivation of EGFR and p-ERK1/2 and PI3KCA mutations. Dual-resistant cells (SKLTR) also showed hyperactivation of HER4 and recovered levels of p-AKT compared with mono-resistant cells. mTOR, p-mTOR and FASN expression remained stable in SKTR, SKLR and SKLTR. In vitro, anti-FASN compounds plus pertuzumab showed synergistic interactions in lapatinib- and dual-resistant cells and improved the results of pertuzumab plus trastuzumab co-treatment. FASN inhibitors combined with temsirolimus displayed the strongest synergistic interactions in resistant cells. In vivo, both orthoxenopatients showed strong response to the antitumor activity of the combination of EGCG with pertuzumab or temsirolimus, without signs of toxicity. We showed that the simultaneous blockade of FASN and HER2 pathways is effective in cells and in breast cancer models refractory to anti-HER2 therapie

Transcriptional upregulation of HER2 expression in the absence of HER2 gene amplification results in cetuximab resistance that is reversed by trastuzumab treatment

The recent identification of HER2 gene amplification as a novel predictor of resistance to the EGFR (HER2)-targeted antibody cetuximab and of response to combination therapies against EGFR and HER2 in wild-type KRAS tumor settings may represent a further step toward personalized medicine for patients with colorectal cancer. Herein, we show that transcriptional upregulation of HER2 expression in the absence of HER2 gene amplification is a molecular phenomenon that takes place in EGFR-dependent, wild-type KRAS squamous cell carcinoma (SCC) cells that acquire resistance to cetuximab. Since cetuximab activity against cetuximab-refractory SCC cells can be fully restored in the presence of the anti-HER2 monoclonal antibody trastuzumab, our findings suggest that, beyond HER2 gene amplification, we might need to redefine the threshold values for HER2 positivity to improve the accuracy of the selection of cetuximab-refractory patients with wild-type KRAS that may benefit from receiving a cetuximab/trastuzumab combinatio