Circumvention Of Resistance Research Articles

Abstract P3-07-16: Pre-clinical study of amcenestrant and HER2-targeted therapies in HER2+/ER+ breast cancer cell line models

Abstract Background: Up to 70% of HER2 over-expressing (HER2+) breast cancers also express estrogen receptor (ER). Dual targeting of HER2 and ER has shown pre-clinical and clinical benefit in HER2+/ER+ breast cancer. Rationale for these combinations is based on circumvention of resistance mediated by crosstalk between HER2 and ER signalling pathways. Fulvestrant is the only selective estrogen receptor degrader (SERD) approved by the FDA for the treatment of ER+ breast cancer. Development of alternative orally available SERDs with improved bioavailability, such as amcenestrant, is underway. The HER2-targeted tyrosine kinase inhibitors (TKIs) lapatinib, neratinib and tucatinib and the antibody drug conjugate T-DM1 are approved for the treatment of HER2+ breast cancer. This pre-clinical study aims to investigate the potential of combining the oral SERD amcenestrant with HER2-targeted therapies for the treatment of HER2+/ER+ breast cancer through identification of synergistic combinations. Methods: ER and HER2 expression levels were assessed in the HER2+/ER+ BT-474 and MDA-MB-361 cell lines by western blot. 5-day acid phosphatase-based assays were used to assess cell viability following treatment with neratinib, lapatinib, tucatinib, and T-DM1; alone and in combination with amcenestrant. All TKIs were commercially sourced, T-DM1 was obtained from Saint Vincent’s University Hospital. Amcenestrant was supplied by Sanofi. IC50 values were determined using Calcusyn, while matrix combination assays of the HER2-targeted therapies combined with amcenestrant were analysed using Combenefit software employing the Loewe model (LM) to determine synergy (Score > 0) or antagonism (Score < 0). All data are the result of a minimum of three independent experiments. Results: HER2 and ER expression was confirmed in the MDA-MB-361 and BT-474 cell lines. MDA-MB-361 cells express higher levels of ER and lower levels of HER2 and phosphorylated HER2 than BT474 cells. MDA-MB-361 exhibited a higher sensitivity to 1 µM amcenestrant alone (% cell viability = 53.3 ± 2.3 %) than the BT-474 (% viability = 85.3 ± 5%). A comparison of IC50 values for each TKI alone or in combination with 1 µM amcenestrant showed amcenestrant significantly lowered the IC50 values for neratinib and tucatinib in BT-474, and all three TKIs in MDA-MB-361 (Table 1). In matrix combination assays, all TKIs exhibited synergistic combinations, with the combination of neratinib and amcenestrant demonstrating the highest synergy values in MDA-MB-361 (LM max value = 25 ± 7) and in BT-474 (LM max value = 19 ± 7). The combination of amcenestrant and T-DM1 displayed additivity (LM value = 0) rather than synergy in the BT-474 cell line, likely due to the high potency of T-DM1 as a single agent. Conclusions: The combination of amcenestrant and HER2-targeting therapies such as neratinib shows potential as a dual-target therapeutic strategy for the treatment of HER2+/ER+ breast cancer and warrants further investigation. Table 1. IC50 values for TKIs alone and with the addition of 1 µM amcenestrant. Significance of chance in IC50 point calculated by Student’s T test, ns = p > 0.05, * = p < 0.05, ** = p < 0.01, SD = standard deviation Citation Format: Amira F. Mahdi, Niall Ashfield, Neil T. Conlon, John Crown, Denis M. Collins. Pre-clinical study of amcenestrant and HER2-targeted therapies in HER2+/ER+ breast cancer cell line models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-16.

Effects of hsa-miR-9-3p and hsa-miR-9-5p on Topoisomerase IIβ Expression in Human Leukemia K562 Cells with Acquired Resistance to Etoposide.

DNA topoisomerase IIα (TOP2α/170; 170 kDa) and topoisomerase IIβ (TOP2β/180; 180 kDa) are targets for a number of anticancer drugs, whose clinical efficacy is attenuated by chemoresistance. Our laboratory selected for an etoposide-resistant K562 clonal subline designated K/VP.5. These cells exhibited decreased TOP2α/170 and TOP2β/180 expression. We previously demonstrated that a microRNA-9 (miR-9)-mediated posttranscriptional mechanism plays a role in drug resistance via reduced TOP2α/170 protein in K/VP.5 cells. Here, it is hypothesized that a similar miR-9 mechanism is responsible for decreased TOP2β/180 levels in K/VP.5 cells. Both miR-9-3p and miR-9-5p are overexpressed in K/VP.5 compared with K562 cells, demonstrated by microRNA (miRNA) sequencing and quantitative polymerase chain reaction. The 3'-untranslated region (3'-UTR) of TOP2β/180 contains miRNA recognition elements (MRE) for both miRNAs. Cotransfection of K562 cells with a luciferase reporter plasmid harboring TOP2β/180 3'-UTR plus miR-9-3p or miR-9-5p mimics resulted in statistically significant decreased luciferase expression. miR-9-3p and miR-9-5p MRE mutations prevented this decrease, validating direct interaction between these miRNAs and TOP2β/180 mRNA. Transfection of K562 cells with miR-9-3p/5p mimics led to decreased TOP2β protein levels without a change in TOP2β/180 mRNA and resulted in reduced TOP2β-specific XK469-induced DNA damage. Conversely, K/VP.5 cells transfected with miR-9-3p/5p inhibitors led to increased TOP2β/180 protein without a change in TOP2β/180 mRNA and resulted in enhancement of XK469-induced DNA damage. Taken together, these results strongly suggest that TOP2β/180 mRNA is translationally repressed by miR-9-3p/5p, that these miRNAs play a role in acquired resistance to etoposide, and that they are potential targets for circumvention of resistance to TOP2-targeted agents. SIGNIFICANCE STATEMENT: Results presented here indicate that miR-9-3p and miR-9-5p play a role in acquired resistance to etoposide via decreased DNA topoisomerase IIβ 180 kDa protein levels. These findings contribute further information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. In addition, miR-9-3p and miR-9-5p overexpression in cancer chemoresistance may lead to future validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy.

Plant-Based Bioinsecticides for Mosquito Control: Impact on Insecticide Resistance and Disease Transmission.

Simple SummaryMosquito-borne diseases cause millions of deaths each year. There has been an increase in the use of insecticides to combat disease transmission caused by mosquitoes. Synthetic insecticides have been effectively used to protect humans from mosquito bites through insecticide-treated mosquito nets, fabrics, and indoor sprays. Despite the considerable progress made in reducing mosquito borne diseases, extensive usage of insecticides has caused serious health problems to humans and animals, insecticide resistance or insensitivity in mosquitoes, and environmental damage. A success in the fight with mosquito disease transmission can only be accomplished by adequate and effective implementation of insecticide resistance monitoring and management programs globally. For this purpose, extensive research focuses on exploring insecticide resistance mechanisms in mosquitoes and how they get resistant to chemical applications over time. The search also focuses on novel compounds that are more effective, safer, and eco-friendly for improved management of mosquito vectors. In this review, we provide the current literature on the synthetic insecticides and how mosquitoes develop resistance to them, with further emphasis on bioinsecticides that could replace conventional synthetic insecticides. In this context, plant-based compounds are explained in detail with their potential applications to control mosquitoes.The use of synthetic insecticides has been a solution to reduce mosquito-borne disease transmission for decades. Currently, no single intervention is sufficient to reduce the global disease burden caused by mosquitoes. Problems associated with extensive usage of synthetic compounds have increased substantially which makes mosquito-borne disease elimination and prevention more difficult over the years. Thus, it is crucial that much safer and effective mosquito control strategies are developed. Natural compounds from plants have been efficiently used to fight insect pests for a long time. Plant-based bioinsecticides are now considered a much safer and less toxic alternative to synthetic compounds. Here, we discuss candidate plant-based compounds that show larvicidal, adulticidal, and repellent properties. Our discussion also includes their mode of action and potential impact in mosquito disease transmission and circumvention of resistance. This review improves our knowledge on plant-based bioinsecticides and the potential for the development of state-of-the-art mosquito control strategies.

Abstract LB131: Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells

Abstract Aim: We investigated the mechanisms driving the concomitant overexpression of ABCG2 and CD133 in cancer stem cells (CSCs) and examined the effect of modulating the underlying machinery on anticancer drug response. Background: Multidrug resistance is a major obstacle to chemotherapy. It is usually associated with increased efflux of anticancer drugs by ATP-binding cassette transporters including ABCG2. ABCG2 is also a key survival factor for CSCs, which lead to recurrence. CSCs have been identified in patient tumor specimens and in cancer cell lines. The CSC hypothesis suggests that anticancer therapies have to target and destroy all residual CSCs in order to produce a durable response. CD133 is a well-characterized stem cell biomarker and it plays crucial role in the self-renewal and tumorigenesis of CSCs. Importantly, ABCG2 and CD133 are co-expressed in CSCs, yet the mechanism(s) driving their concomitant high expression remain elusive. mRNA polyadenylation is a key step regulating the maturation of eukaryotic mRNAs by adding a poly A tail to their 3' ends. Alternative polyadenylation, commonly observed in cancer cells, is a phenomenon responsible for the production of multiple mRNA isoforms bearing different length of 3'-untranslated region (3'UTR). Method: Sub-population of cancer cells from three ABCG2-overexpressing multidrug resistant colon cancer cell lines were sorted by Hoechst dye exclusion or CSC markers (DCLK1+LGR5+BMI1+), respectively, as side population (SP) and putative CSC cells. Expression of ABCG2, CD133, and a few master mRNA polyadenylation trans-acting regulators was compared in SP (or CSC) versus non-SP (or non-CSC) cells. Altered 3'UTR isoforms of ABCG2 and CD133 were examined by 3'RACE assay and 3'UTR isoform specific PCR. The anticancer activity of common chemotherapeutic drugs was examined after genetic modulation of the key mRNA polyadenylation regulators identified. Result: The mRNA stability and expression of both ABCG2 and CD133 were found to be remarkably higher in SP cells (or CSC cells) than in non-SP cells (or non-CSC cells). By 3'RACE assay, shortening of ABCG2 and CD133 3'UTR was evidenced in SP (and CSC) cells but not in non-SP (and non-CSC) cells, thereby increasing mRNA stability and/or translation by escaping the repression from regulatory elements found on long mRNA 3'UTR. Expression of two master mRNA polyadenylation regulators (PABPN1 and CSTF2) was found to be remarkably lower and higher, respectively, in SP (and CSC) than in non-SP (and non-CSC) cells, presumably allowing the preferential utilization of proximal 3'UTR to mediate high expression of ABCG2 and CD133. Genetic silencing of CSTF2 was shown to enhance the anticancer effect of SN-38 and reduce the colony formation capacity of CSC cells. Conclusion: A novel mRNA 3'UTR shortening machinery was shown to mediate the simultaneous overexpression of ABCG2 and CD133 in SP and CSC cells. It may represent useful drug target for circumvention of resistance and eradication of CSCs. Citation Format: Kenneth K.W. To. Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB131.

PPARgamma agonists sensitize PTEN-deficient resistant lung cancer cells to EGFR tyrosine kinase inhibitors by inducing autophagy

We aimed to develop novel drug combination strategy to overcome drug resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in the treatment of non-small cell lung cancer (NSCLC). Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor, which upon activation upregulates phosphatase and tensin homolog (PTEN) to inhibit cell signaling downstream of PI3K to mediate apoptosis. To this end, PTEN loss is a known mechanism contributing to resistance to EGFR TKIs. Therefore, PPARγ agonists are hypothesized to overcome EGFR TKI resistance. Using human NSCLC cell models with PTEN deficiency, the potentiation of EGFR TKI anticancer activity by PPARγ agonists was evaluated. PPARγ agonists were found to upregulate PTEN, subsequently inhibiting the PI3K-Akt signaling pathway, and thus enhancing the anticancer activity of gefitinib (a first generation EGFR TKI). Chemical and genetic inhibition of PPARγ were shown to prevent this potentiation of anticancer activity by PPARγ agonists, thus confirming the crucial role played by PPARγ activation. Interestingly, the tested PPARγ agonists were also found to induce autophagy, as evidenced by the increased expression of an autophagy marker LC3-II and the autophagic degradation of p62/SQSTM1. PPARγ agonists-induced autophagic cell death was believed to contribute to the circumvention of resistance in PTEN-deficient cells because the genetic silencing of ATG5 (an autophagy mediator) was found to eliminate the drug potentiation effect by the PPARγ agonists. Our findings thus provide the basis for the rational and personalized use of PPARγ agonists in combination with EGFR TKIs in lung cancer patients.

CUDC-907, a dual HDAC and PI3K inhibitor, reverses platinum drug resistance.

Platinum (Pt)-based anticancer drugs are the mainstay of treatment for solid cancers. However, resistance to Pt drugs develops rapidly, which can be caused by overexpression of multidrug resistance transporters and activation of DNA repair. CUDC-907 is a potent molecular targeted anticancer agent, rationally designed to simultaneously inhibit histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K). We investigated the potentiation effect of CUDC-907 on Pt drugs in resistant cancer cells. ABCC2 stably-transfected HEK293 cells and two pairs of parental and Pt-resistant cancer cell lines were used to test for the circumvention of resistance by CUDC-907. Chemosensitivity was assessed by the sulphorhodamine B assay. Drug combinations were evaluated by the median effect analysis. ABCC2 transport activity was examined by flow cytometric assay. Cellular Pt drug accumulation and DNA platination were detected by inductively coupled plasma optical emission spectroscopy. ABCC2, ERCC1 and p21 expression were evaluated by quantitative real-time PCR. Cell cycle analysis and apoptosis assay were performed by standard flow cytometric method. The combination of CUDC-907 with cisplatin were found to exhibit synergistic cytotoxic effect in cisplatin-resistant cancer cells. In Pt-resistant cancer cells, CUDC-907 apparently circumvented the resistance through inhibition of ABCC2 and DNA repair but induction of cell cycle arrest. In the presence of CUDC-907, cellular accumulation of Pt drugs and formation of DNA-Pt adducts were found to be increased whereas expression levels of ABCC2 and ERCC1 was inhibited in Pt-resistant cells. The data advocates further development of CUDC-907 as a resistance reversal agent for use in combination cancer chemotherapy.

Abstract LB-126: PPARgamma agonists sensitize PTEN-deficient lung cancer cells resistant to EGFR tyrosine kinase inhibitors

Abstract We aimed to develop novel drug combination strategy to overcome drug resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in the treatment of lung cancer. EGFR TKIs are approved for treating advanced lung cancer that harbors the sensitizing EGFR mutations. However, resistance to EGFR TKIs is almost inevitable for all patients due to multifactorial mechanisms. The PI3K/Akt/mTOR pathway is a major signaling pathway regulated by EGFR to drive cancer survival. Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor, which upon activation upregulates PTEN to inhibit cell signaling downstream of PI3K to mediate apoptosis. To this end, PTEN loss is a known mechanism contributing to intrinsic resistance to EGFR TKIs. Therefore, PPARγ agonists are hypothesized to overcome EGFR TKI resistance. Using well defined human non-small cell lung cancer (NSCLC) cell model with PTEN deficiency, the potentiation of EGFR TKI anticancer activity by PPARγ agonists was demonstrated. PPARγ agonists were found to upregulate PTEN, subsequently inhibit the PI3K-Akt signaling pathway, and thus enhancing the anticancer activity of gefitinib (a first generation EGFR TKI). Specific PPARγ inhibitor (GW9662) and genetic silencing of PPARγ were shown to prevent this potentiation of anticancer activity by PPARγ agonists, thus confirming the crucial role played by PPARγ activation. Interestingly, the tested PPARγ agonists were also found to induce autophagy, as evidenced by the increased expression of an autophagy marker LC3-II and the autophagic degradation of p62/SQSTM1. PPARγ agonists-induced autophagic cell death was believed to contribute to the circumvention of resistance in PTEN-deficient cells because the genetic silencing of ATG5 (an autophagy mediator) was found to eliminate the drug potentiation effect by the PPARγ agonists. Our findings thus provide the basis for the rational and personalized use of PPARγ agonists in combination with EGFR TKIs in lung cancer patients with specific resistance mechanism. Citation Format: Kenneth KW To, Wing Sum Tong, William KK Wu, Herbert HF Loong. PPARgamma agonists sensitize PTEN-deficient lung cancer cells resistant to EGFR tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-126. doi:10.1158/1538-7445.AM2017-LB-126

Pesticide Chemical Research in Toxicology: Lessons from Nature.

Pesticide researchers are students of nature, and each new compound and mechanism turns a page in the ever-expanding encyclopedia of life. Pesticides are both probes to learn about life processes and tools for pest management to facilitate food production and enhance health. In contrast to some household and industrial chemicals, pesticides are assumed to be hazardous to health and the environment until proven otherwise. About a thousand current pesticides working by more than 100 different mechanisms have helped understand many processes and coupled events. Pesticide chemical research is a major source of toxicology information on new natural products, novel targets or modes of action, resistance mechanisms, xenobiotic metabolism, selective toxicity, safety evaluations, and recommendations for safe and effective pest management. Target binding site models help define the effect of substituent changes and predict modifications for enhanced potency and safety and circumvention of resistance. The contribution of pesticide chemical research in toxicology is illustrated here with two each of the newer or most important insecticides, herbicides, and fungicides. The insecticides are imidacloprid and chlorantraniliprole acting on the nicotinic acetylcholine receptor and the ryanodine receptor Ca2+ channel, respectively. The herbicides are glyphosate that inhibits aromatic amino acid biosynthesis and mesotrione that prevents plastoquinone and carotenoid formation. The fungicides are azoxystrobin inhibiting the Qo site of the cytochrome bc1 complex and prothioconazole inhibiting the 14α-demethylase in ergosterol biosynthesis. The two target sites involved for each type of pesticide account for 27-40% of worldwide sales for all insecticides, herbicides, and fungicides. In each case, selection for resistance involving a single amino acid change in the binding site or detoxifying enzyme circumvents the pesticide chemists's structure optimization and guarantees survival of the pest and a continuing job for the design chemist. These lessons from nature are a continuing part of pest management and maintaining human and environmental health.

Data showing the circumvention of oxaliplatin resistance by vatalanib in colon cancer.

We have recently reported that vatalanib, an orally active small molecule multi-tyrosine kinase inhibitor (Hess-Stumpp et al., 2005 [1]), can sensitize multidrug resistant (MDR) colon cancer cells to chemotherapy under hypoxia by inhibiting two MDR transporters ABCB1 and ABCG2 (To et al., 2015 [2]). This data article describes the possible circumvention of resistance to specifically platinum (Pt)-based anticancer drugs by vatalanib via inhibition of two other efflux transporters ABCC2 and ATP7A. Data from the flow cytometric transporter efflux assay showed specific inhibition of ABCC2 activity by vatalanib in stable transfected cells and ABCC2-overexpressing oxaliplatin-resistant colon cancer cells HCT116/Oxa. We also performed the transporter ABCC2 ATPase assay and showed an increase in ATP hydrolysis by ABCC2 in the presence of vatalanib. ATP7A mRNA expression was also shown to be upregulated in HCT116/Oxa cells. Vatalanib was shown to suppress this upregulated ATP7A expression. Data from the cellular Pt accumulation assay showed a lower Pt accumulation in HCT116/Oxa cells than the parental sensitive HCT116 cells. Vatalanib was shown to increase cellular Pt accumulation in a concentration-dependent manner. Combination of oxaliplatin and vatalanib was shown to restore the suppressed apoptosis in HCT116/Oxa cells.

Circumvention of resistance to photodynamic therapy in doxorubicin-resistant sarcoma by photochemical internalization of gelonin

A wide range of anti-cancer therapies have been shown to induce resistance upon repetitive treatment and such adapted resistance may also cause cross-resistance to other treatment modalities. We here show that MES-SA/Dx5 cells with adapted resistance to doxorubicin (DOX) are cross-resistant to photodynamic therapy (PDT). A DOX-induced increased expression of the reactive oxygen species (ROS)-scavenging proteins glutathione peroxidase (GPx) 1 and GPx4 in MES-SA/Dx5 cells was indicated as the mechanism of resistance to PDT in line with the reduction in PDT-generated ROS observed in this cell line. ROS-induced p38 activation was, in addition, shown to be reduced to one-third of the signal of the parental MES-SA cells 2h after PDT, and addition of the p38 inhibitor SB203580 confirmed p38 activation as a death signal after PDT in the MES-SA cells. The MES-SA/Dx5 cells were also cross-resistant to ionizing radiation in agreement with the increased GPx1 and GPx4 expression. Surprisingly, PDT-induced endo/lysosomal release of the ribosome-inactivating protein gelonin (photochemical internalization (PCI)) was more effective in the PDT-resistant MES-SA/Dx5 cells, as measured by synergy calculations in both cell lines. Analysis of death-inducing signaling indicated a low activation of caspase-3 and a strong PARP I cleavage after PDT and PCI in both cell lines. The PARP I activation was, however, stronger after PCI than after PDT in the MES-SA cells, but not in the MES-SA/Dx5 cells, and therefore cannot explain the strong PCI effect in the MES-SA/Dx5 cells. In conclusion PCI of recombinant gelonin circumvents ROS resistance in an apoptosis-independent manner.

NF-κB is involved in SHetA2 circumvention of TNF-α resistance, but not induction of intrinsic apoptosis

Treatment of cancer with tumor necrosis factor-alpha (TNF-alpha) is hindered by resistance and toxicity. The flexible heteroarotinoid, SHetA2, sensitizes resistant ovarian cancer cells to TNF-alpha-induced extrinsic apoptosis, and also induces intrinsic apoptosis as a single agent. This study tested the hypothesis that nuclear factor-kappaB (NF-kappaB) is involved in SHetA2-regulated intrinsic and extrinsic apoptosis. SHetA2 inhibited basal and TNF-alpha-induced or hydrogen peroxide-induced NF-kappaB activity through counter-regulation of upstream kinase (IkappaB kinase) activity, inhibitor protein (IkappaB-alpha) phosphorylation, and p-65 NF-kappaB subunit nuclear translocation, but independently of reactive oxygen species generation. Ectopic over-expression of p-65, or treatment with TNF-alpha receptor 1 (TNFR1) small interfering RNA or a caspase-8 inhibitor, each attenuated synergistic apoptosis by SHetA2 and TNF-alpha, but did not affect intrinsic apoptosis caused by SHetA2. In conclusion, NF-kappaB repression is involved in SHetA2 circumvention of resistance to TNF-alpha-induced extrinsic apoptosis, but not in SHetA2 induction of intrinsic apoptosis.

Repression of the BH3-only molecule PMAIP1/Noxa impairs glucocorticoid sensitivity of acute lymphoblastic leukemia cells

Glucocorticoid (GC)-induced apoptosis plays a major role in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Members of the BCL2 family of pro- and anti-apoptotic proteins are regulated by GC, but to what extent these regulations contribute to GC-induced cell death and resistance development is poorly understood. Using primary lymphoblasts from ALL children during systemic GC monotherapy and related cell lines, we have previously shown that the response of the BCL2 rheostat to GC was dominated by induction of the pro-apoptotic BH3-only molecules BMF and BCL2L11/Bim, but we also observed an unexpected significant repression of the pro-apoptotic BCL2 protein PMAIP1/Noxa. Here, we report that GC represses Noxa mRNA levels and also interferes with its protein stability in a proteasome-dependent manner. Prevention of GC-mediated Noxa repression by conditional expression of transgenic Noxa changed the kinetics of GC-induced apoptosis to resemble cell death induced by BimEL alone. Hence, GC appear to activate functionally relevant pro- as well as anti-apoptotic pathways in ALL cells. Interfering with the anti-apoptotic component of the GC response might contribute to improved therapeutic approaches and circumvention of resistance to this therapy.

Molecular mechanisms of antimony resistance in Leishmania

Leishmaniasis causes significant morbidity and mortality worldwide. The disease is endemic in developing countries of tropical regions, and in recent years economic globalization and increased travel have extended its reach to people in developed countries. In the absence of effective vaccines and vector-control measures, the main line of defence against the disease is chemotherapy. Organic pentavalent antimonials [Sb(V)] have been the first-line drugs for the treatment of leishmaniasis for the last six decades, and clinical resistance to these drugs has emerged as a primary obstacle to successful treatment and control. A multiplicity of resistance mechanisms have been described in resistant Leishmania mutants developed in vitro by stepwise increases of the concentration of either antimony [Sb(III)] or the related metal arsenic [As(III)], the most prevalent mechanism being upregulated Sb(III) detoxification and sequestration. With the availability of resistant field isolates, it has now become possible to elucidate mechanisms of clinical resistance. The present review describes the mechanisms of antimony resistance in Leishmania and highlights the links between previous hypotheses and current developments in field studies. Unravelling the molecular mechanisms of clinical resistance could allow the prevention and circumvention of resistance, as well as rational drug design for the treatment of drug-resistant Leishmania.

Drug resistance in ovarian cancer: The emerging importance of gene transcription and spatio-temporal regulation of resistance

Resistance to carboplatin plus paclitaxel, one of the most active drug combinations in ovarian cancer, is the major barrier to the successful long-term treatment of this disease. Understanding the mechanisms involved is a first step towards rational strategies to overcome drug resistance and is an area of intense research effort. Recent work has identified several gene families which appear to contribute to the evolution of drug resistance and which are involved in regulating DNA damage, apoptosis and survival signalling. These genes may be co-ordinately regulated as part of a gene expression program that confers drug resistance through multiple pathways. The subcellular localisation of the gene products and their kinetic regulation following exposure to chemotherapeutic agents may also play a part in the development of drug resistance. This provides a more complex paradigm for drug resistance in which the steady-state expression of a single gene may not be predictive of response to therapy. Nevertheless, the identification of critical genes, most relevant to the development of clinical drug resistance, is now feasible through microarray analysis of tumour samples, and strategies aimed at the circumvention of resistance can be developed using these data.

DNA interactions of new antitumor platinum complexes with trans geometry activated by a 2-metylbutylamine or sec-butylamine ligand

The global modification of mammalian and plasmid DNAs by novel platinum compounds, trans-[PtCl 2(NH 3)(Am)], where Am=2-methylbutylamine or sec-butylamine was investigated in cell-free media using various biochemical and biophysical methods. These modifications were analyzed in the context of the activity of these new compounds in several tumor cell lines including those resistant to antitumor cis-diamminedichloroplatinum(II) (cisplatin). The results showed that the replacement of one amine group by 2-methylbutylamine or sec-butylamine ligand in clinically ineffective trans-diamminedichloroplatinum(II) (transplatin) resulted in a radical enhancement of its activity in tumor cell lines so that they are more cytotoxic than cisplatin and exhibited significant antitumor activity including activity in cisplatin-resistant tumor cells. Importantly, this replacement also markedly altered DNA binding mode of transplatin and reduced the efficiency of repair systems to remove the adducts of the new analogues from DNA. The results support the view that one strategy to activate trans geometry in bifunctional platinum(II) compounds including circumvention of resistance to cisplatin may consist in a chemical modification of the ineffective transplatin which results in an increased efficiency to form DNA interstrand cross-links.

Chromosomal imbalance maps of human 5FU-resistant colorectal cancer cell lines: implications in the analysis of 5FU-acquired resistance mechanisms.

Thymidylate synthase (TS), a critical enzyme in the de novo synthesis of thymidylate, is an important target for fluoropyrimidines and folate-based TS inhibitors. Overexpression of TS has been correlated to 5-fluorouracil (5FU)-resistance. Because 5FU still remains a basic component of the treatment of colorectal cancer, circumvention of resistance is of vital importance. A panel of sensitive (HT29 and LoVo) and 5FU-resistant colorectal cancer cell lines (HT29-5FUR and LoVo-5FUR) were subjected to comparative genomic hybridization (CGH) analysis to identify possible amplified/deleted regions associated with 5FU-resistance in colon tumours. We have identified chromosomal gains at 5p, 6, 7p, 7q and 8q and one loss at 3q in 5FU-resistant cells as compared to corresponding sensitive cell lines. Neither chromosomal gains at 18p nor gene amplification of TS were observed in our resistant cell lines although an overexpression of TS gene exists (at mRNA level) in these cell lines as compared with corresponding parental cells. Most of the chromosomal gains identified in this study occur frequently in sporadic colorectal tumours and has been associated to a poor prognosis and a greater progression of the tumour and could be related to a worse chemotherapy response. The chromosomal imbalance profile detected in 5FU-resistant cell lines should provide a basis for interpreting mechanisms of 5FU-resistance in colorectal cancer and also possibly in other tumours treated with this agent. This study also identified new genes potentially implicated in 5FU-resistance and suggests new targets that could be useful for the chemotherapy treatment of colorectal cancer.

DNA binding mode of the cis and trans geometries of new antitumor nonclassical platinum complexes containing piperidine, piperazine, or 4-picoline ligand in cell-free media. Relations to their activity in cancer cell lines.

The global modification of mammalian and plasmid DNAs by novel platinum compounds, cis- or trans-[PtCl(2)(NH(3))(Am)], where Am = NH(3), nonplanar heterocycle piperidine, piperazine, or aromatic planar heterocycle 4-picoline, was investigated in cell-free media using various biochemical and biophysical methods. These modifications have been compared with the activity of these new compounds in several tumor cell lines including those resistant to antitumor cis-diamminedichloroplatinum(II) (cisplatin). The results show that the replacement of the NH(3) group in cisplatin by the heterocyclic ligands does not considerably affect the DNA binding mode of this drug. Cytotoxicity studies have revealed that the replacement lowers the activity of the platinum compound in both sensitive and resistant cell lines. It has been suggested that the reduced activity of these analogues of cisplatin is associated with some features of the damaged DNA and/or its cellular processing. Alternatively, the reduced activity of the analogues of cisplatin might also be due to the factors that do not operate directly at the level of the target DNA, such as intracellular platinum uptake. In contrast to the analogues of cisplatin, the replacement of one ammine group by the heterocyclic ligand in its clinically ineffective trans isomer (transplatin) results in a radical enhancement of its activity in tumor cell lines. Importantly, this replacement also markedly alters the DNA binding mode of transplatin. The results support the view that one strategy of how to activate the trans geometry in bifunctional platinum(II) compounds including circumvention of resistance to cisplatin may consist of a chemical modification of the ineffective transplatin that results in an increased stability of its intrastrand cross-links in double-helical DNA and/or in an increased efficiency to form interstrand cross-links.

Apoptosis is induced in both drug-sensitive and multidrug-resistant hepatoma cells by somatostatin analogue TT-232.

Clinical resistance to chemotherapeutic drugs is an important problem in the treatment of cancer; the circumvention of resistance has become one of the basic goals of cancer therapy. The most frequent form of primary liver cancer is hepatocellular carcinoma, which is essentially refractory to chemotherapy. We earlier showed that TT-232, a new somatostatin analogue developed in our laboratory, exerted a strong antiproliferative effect both in vitro and in vivo, but no growth hormone release inhibitory or antisecretory activity. Here we report that TT-232 has a pronounced antiproliferative effect on differentiated and dedifferentiated, drug-sensitive and multidrug-resistant hepatocellular carcinoma cell lines. TT-232 induces apoptosis at comparable levels in all these hepatoma variants demonstrating that the multidrug resistance of hepatomas does not correlate with a reduced susceptibility to apoptosis induction. These results clearly reveal that the machinery involved in apoptosis is functional in both drug-sensitive and resistant hepatoma variants and can be activated by the somatostatin analogue TT-232. © 1999 Cancer Research Campaign

Folate-based thymidylate synthase inhibitors in cancer chemotherapy.

Understanding the relationship between chemical structure and biological properties of folate analogs, particularly their interactions with the target enzymes, transport proteins and folate-metabolizing enzyme, folylpolyglutamate synthetase (FPGS), has enabled the rational design and development of the selective thymidylate synthase (TS) inhibitors with folate-based structures for clinical uses. These compounds specifically inhibit TS devoid of concomitant effects at other loci, unlike 5-fluorouracil (5-FU). ZD1694 ('Tomudex') was designed as a non-nephrotoxic and highly active analog of N10-propargyl-5,8-dideazafolic acid (CB3717), which is a potent TS inhibitor but had unacceptable nephrotoxicity caused by its poor water solubility. The potent cytotoxic activity of ZD1694 is dependent upon active uptake into cells via the reduced folate carrier (RFC), and subsequent rapid and extensive metabolism to polyglutamate forms inside cells. Marked enhancement of the TS inhibitory activity has been noted as the glutamate chain is elongated. Polyglutamation is critical to the biological activity of ZD1694 against tumor and normal proliferating tissues. The retentive property of ZD1694 polyglutamates inside cells led to a single, infrequent administration schedule in clinical studies. ZD1694 has completed phase I and phase II evaluation with activity observed in several tumor types, particularly in colorectal cancer with a 26% objective response rate. A recent European phase III study of ZD1694, randomized against a 5-FU plus leucovorin regimen, demonstrated an equivalent response rate for advanced colorectal cancer (complete or partial responses; 20 versus 17%) and less toxicity than seen with the latter regimen. The newer selective TS inhibitors, which retain potency for TS inhibition but are not substrates for RFC and/or FPGS, are currently under clinical evaluation. These classes of compound may have benefits for circumvention of resistance by virtue of alterations in these protein functions and for the management of toxicity.

305 Circumvention of resistance to 5-fluorouracil by schedule-oriented biochemical modulation in advanced colon cancer patients

We have recently demonstrated that bolus FUra treatments of human colon carcinoma cells, HCT-8, produce resistance via an RNA-related mechanism, while prolonged exposures to the fluoropyrimidine is responsible for a DNA-related mechanism of resistance. In addition, cells resistant to short term FUra exposure retain full sensitivity to the continuous exposure to the same agent. These data suggest that biochemical modulation of FUra should take into account the schedule of fluoropyrimidine administration. Based on this rationale, we completed a phase 2 trial of schedule-oriented biochemical modulation of FUra in advanced colorectal cancer patients, based upon a hybrid regimen of 2 biweekly-cycles of FUra bolus (600 mg/sqm), preceeded by (24 h interval) MTX, 200 mg/sqm (in order to maximize the RNA effect of the drug) alternating with FUra continuous infusion, 200 mg/sqm daily for 3 weeks, modulated by leucovorin, 20 mg/sqm weekly bolus (in order to maximize the DNA effect). Among the 33 consecutive patients accrued there were 3 CR and 13 PR (RR = 48%, 95% CL, 31–66%). Eleven patients had a minor response and 4 of them showed tumor shrinkage ranging between 46% and 49%. After a median follow-up time of 26 months, 10 patients are still alive. The median PFS and overall S were 9.6 and 20.2 months, respectively. The low toxicity of the bolus part of our regimen prompted us to pursue its intensification by adding a further modulator to MTX. Our recent finding of a strong synergism between bolus FUra and IFN, obtained in vitro on the same tumor model, generated a phase 2 trial employing the same regimen plus IFN (3,000,000 U im q12 h × 4, starting at the time of FUra bolus administration). Among 42 patients 4 CR, 15 PR (RR = 45%), 6 MR and 10 SD were obtained. Since the results of the two trials are similar showing twice as much activity and less toxicity than bolus FUra + LV or MTX → FUra, a randomized comparison is now ongoing between our original hybrid regimen without IFN and MTX → FUra.