10 research outputs found
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), [(Ρ 6-bip)Os(4-CO 2-pico)Cl] (bip = biphenyl, pico = picolinate) (4), [(Ρ 6-p-cym)RuCl(dap)] + (p-cym = p-cymene) (5), and [(Ρ 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 ¹ 2 Ο in MCF-7 cells and IC 50 = 26 ¹ 3 Ο 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 ¹ 2.6 Ο 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. Š 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 [(Ρ6-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,Nâ˛-di-Boc-Nâł-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 â 80 ÎźM), whereas the neamine conjugate (4) was inactive (IC50 â 200 ÎźM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 ÎźM in DU-145 and IC50 = 11.33 ÎźM 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 ÎźM 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 ÎźM 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
AnĂ lisi de la vehiculitzaciĂł de compostos metĂ l¡lics com a agents antitumorals i de la famĂlia 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 quimioterĂ pia actual tenen un Ăşs limitat com a conseqßència dels efectes adversos que produeixen i de les resistències que desenvolupen les cèl¡lules tumorals a aquests tractaments. En aquesta tesi, sâha contribuĂŻt en el coneixement dâaquestes limitacions a travĂŠs de dos estudis. Dâuna banda, sâha demostrat que la uniĂł de complexos metĂ l¡lics de platĂ, ruteni i osmi a anĂ legs de lâoctreotide i lâĂ cid fòlic tĂŠ un elevat potencial per dirigir aquests conjugats especĂficament cap les cèl¡lules tumorals. Dâaltra banda, sâha provat que un dels mecanismes de resistència implicats en la baixa resposta dels agents dirigits a inhibir la via HER-EGF en el cĂ ncer de pròstata es basa en la sobreexpressiĂł de receptors i lligands de la pròpia via alternatius a lâinhibit; en concret, sâha demostrat que la sobreexpressiĂł i lâactivaciĂł autocrina del receptor HER3 tenen un paper clau en aquest procĂŠ
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 [(Ρ6-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,Nâ˛-di-Boc-Nâł-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 â 80 ÎźM), whereas the neamine conjugate (4) was inactive (IC50 â 200 ÎźM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 ÎźM in DU-145 and IC50 = 11.33 ÎźM 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 ÎźM 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 ÎźM 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 [(Ρ<sup>6</sup>-<i>p</i>-cym)ÂRuClÂ(PPh<sub>3</sub>)<sub>2</sub>]<sup>+</sup>, 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>â˛-di-Boc-<i>N</i>âł-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 desorptionâionization
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 neomycinâruthenium conjugate (<b>2</b>) displayed
moderate antiproliferative activity in both cancer cell lines (IC<sub>50</sub> â 80 ÎźM), whereas the neamine conjugate (<b>4</b>) was inactive (IC<sub>50</sub> â 200 ÎźM). However,
the guanidinylated analogue of the neomycinâruthenium conjugate
(<b>3</b>) required much lower concentrations than the parent
conjugate for equal effect (IC<sub>50</sub> = 7.17 ÎźM in DU-145
and IC<sub>50</sub> = 11.33 ÎźM 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>), IC<sub>50</sub> values indicate that aminoglycoside-containing
conjugates are about 2-fold more cytotoxic in normal cells (e.g.,
IC<sub>50</sub> = 49.4 ÎźM 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 (IC<sub>50</sub> = 12.75 ÎźM 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), guanidinoneomycinâruthenium
conjugate (<b>3</b>) was more cytotoxic to cancer cells (about
1.8-fold), whereas the opposite tendency applied for neomycinâruthenium
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 rutheniumÂ(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 Subtypeâ2 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<sub>2</sub>(dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (<b>3</b>), [(Ρ<sup>6</sup>-bip)ÂOsÂ(4-CO<sub>2</sub>-pico)ÂCl]
(bip = biphenyl, pico = picolinate) (<b>4</b>), [(Ρ<sup>6</sup>-<i>p</i>-cym)ÂRuClÂ(dap)]<sup>+</sup> (<i>p</i>-cym = <i>p</i>-cymene) (<b>5</b>), and [(Ρ<sup>6</sup>-<i>p</i>-cym)ÂRuClÂ(imidazole-CO<sub>2</sub>H)Â(PPh<sub>3</sub>)]<sup>+</sup> (<b>6</b>), were synthesized by using
a solid-phase approach. Conjugates <b>3</b>â<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 (IC<sub>50</sub> = 63 Âą 2 ÎźM
in MCF-7 cells and IC<sub>50</sub> = 26 Âą 3 ÎźM in DU-145
cells) with active participation of somatostatin receptors in cellular
uptake. Similar cytotoxic activity was found in a normal cell line
(IC<sub>50</sub> = 45 Âą 2.6 ÎźM 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