Drug Influx Research Articles

Enhanced anticancer effect of thymidylate synthase dimer disrupters by promoting intracellular accumulation

IntroductionThymidylate synthase (TS) plays a crucial role in cellular growth, proliferation, DNA synthesis, and repair, thus gaining attention for targeted therapies in cancer. TS overexpression and the altered pharmacokinetics of anti-TS drugs are among the most prominent causes of cellular resistance. Decreased drug influx and/or efficient efflux result in reduced drug access to the intracellular targets.ResultsIn this study, we have evaluated and demonstrated the increased cytotoxic efficacy of novel TS dimer disrupters (Ddis) in the presence of specific inhibitors of drug efflux protein pumps in ovarian and colon cancer cells, suggesting that these compounds are substrates of the cellular drug extruders. A second strategy adopted to favor intracellular accumulation was to employ, as a drug delivery system, a molecular tool able to help less lipophilic compounds to cross the cell membrane. The Ddis were delivered through the SAINT-Protein transfection agent. The observed cell-killing effects agreed with the reduction of TS protein level and cell cycle perturbation.ConclusionOverall, this preclinical study suggests that the innovative TS dimer disrupters can be optimized by increasing their intracellular accumulation by both inhibiting their outflow and/or enhancing cellular uptake.

The role of glycolysis in tumorigenesis: From biological aspects to therapeutic opportunities

Glycolytic metabolism generates energy and intermediates for biomass production. Tumor-associated glycolysis is upregulated compared to normal tissues in response to tumor cell-autonomous or non-autonomous stimuli. The consequences of this upregulation are twofold. First, the metabolic effects of glycolysis become predominant over those mediated by oxidative metabolism. Second, overexpressed components of the glycolytic pathway (i.e. enzymes or metabolites) acquire new functions unrelated to their metabolic effects and which are referred to as “moonlighting” functions. These functions include induction of mutations and other tumor-initiating events, effects on cancer stem cells, induction of increased expression and/or activity of oncoproteins, epigenetic and transcriptional modifications, bypassing of senescence and induction of proliferation, promotion of DNA damage repair and prevention of DNA damage, antiapoptotic effects, inhibition of drug influx or increase of drug efflux. Upregulated metabolic functions and acquisition of new, non-metabolic functions lead to biological effects that support tumorigenesis: promotion of tumor initiation, stimulation of tumor cell proliferation and primary tumor growth, induction of epithelial-mesenchymal transition, autophagy and metastasis, immunosuppressive effects, induction of drug resistance and effects on tumor accessory cells. These effects have negative consequences on the prognosis of tumor patients. On these grounds, it does not come to surprise that tumor-associated glycolysis has become a target of interest in antitumor drug discovery. So far, however, clinical results with glycolysis inhibitors have fallen short of expectations. In this review we propose approaches that may allow to bypass some of the difficulties that have been encountered so far with the therapeutic use of glycolysis inhibitors.

Cisplatin Resistance in Cancer Therapy: Causes and Overcoming Strategies

AbstractDr. S. Shruthi, Prof. Dr. K. Bhasker Shenoy. Cisplatin (CDDP) is the first metal‐based drug and has been a popular drug in the treatment of various types of cancer for decades. Cancer cell death is promoted by this platinum‐based drug by generating DNA adducts and activating apoptotic signaling pathways. The development of drug resistance is the foremost clinical concern that compromises its efficacy. The mechanism of CDDP resistance encompasses changes in drug influx and efflux, drug accumulation reduction, enhanced drug detoxification, and repair which may affect its therapeutic value. Researchers have paid significant attention to overcoming CDDP resistance, which provides an opportunity to enhance clinical outcomes. Combinational therapy, use of CDDP analogs, phytochemicals, nanoparticle‐mediated drug delivery, gene targeting and regulation of drug induced epigenetic changes are the specific regimens to overcome CDDP resistance and treatment burden. In this review, we summarize the mechanisms involved in CDDP resistance and strategies considered to overcome these hurdles concerning preclinical and clinical studies. This may provide insights into the further development of novel approaches to uproot CDDP resistance, to ascertain a safer and more effective cancer therapy.

Delivery of miR-29a improves the permeability of cisplatin by downregulating collagen I expression

In the clinical setting, chemotherapy is the most widely used antitumor treatment, however, chemotherapy resistance significantly limits its efficacy. Reduced drug influx is a key mechanism of chemoresistance, and inhibition of the complexity of the tumor microenvironment (TME) may improve chemotherapy drug influx and therapeutic efficiency. In the current study, we identified that the major extracellular matrix protein collagen I is more highly expressed in lung cancer tissues than adjacent tissues in patients with lung cancer. Furthermore, Kaplan–Meier analysis suggested that COL1A1 expression was negatively correlated with the survival time of patients with lung cancer. Our previous study demonstrated that miR-29a inhibited collagen I expression in lung fibroblasts. Here, we investigated the effect of miR-29a on collagen I expression and the cellular behavior of lung cancer cells. Our results suggest that transfection with miR-29a could prevent Lewis lung carcinoma (LLC) migration by downregulating collagen I expression, but did not affect the proliferation, apoptosis, and cell cycle of LLC cells. In a 3D tumoroid model, we demonstrated that miR-29a transfection significantly increased cisplatin (CDDP) permeation and CDDP-induced cell death. Furthermore, neutral lipid emulsion-based miR-29a delivery improved the therapeutic effect of cisplatin in an LLC spontaneous tumor model in vivo. In summary, this study shows that targeting collagen I expression in the TME contributes to chemotherapy drug influx and improves therapeutic efficacy in lung cancer.

Murepavadin induces envelope stress response and enhances the killing efficacies of β-lactam antibiotics by impairing the outer membrane integrity of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that can cause a variety of acute and chronic infections. The bacterium is highly resistant to numerous antibiotics. Murepavadin is a peptidomimetic antibiotic that blocks the function of P. aeruginosa lipopolysaccharide (LPS) transport protein D (LptD), thus inhibiting the insertion of LPS into the outer membrane. In this study, we demonstrated that sublethal concentrations of murepavadin enhance the bacterial outer membrane permeability. Proteomic analyses revealed the alteration of protein composition in bacterial inner and outer membranes following murepavadin treatment. The antisigma factor MucA was upregulated by murepavadin. In addition, the expression of the sigma E factor gene algU and the alginate synthesis gene algD was induced by murepavadin. Deletion of the algU gene reduces bacterial survival following murepavadin treatment, indicating a role of the envelope stress response in bacterial tolerance. We further demonstrated that murepavadin enhances the bactericidal activities of β-lactam antibiotics by promoting drug influx across the outer membrane. In a mouse model of acute pneumonia, the murepavadin-ceftazidime/avibactam combination showed synergistic therapeutic effect against P. aeruginosa infection. In addition, the combination of murepavadin with ceftazidime/avibactam slowed down the resistance development. In conclusion, our results reveal the response mechanism of P. aeruginosa to murepavadin and provide a promising antibiotic combination for the treatment of P. aeruginosa infections.IMPORTANCEThe ever increasing resistance of bacteria to antibiotics poses a serious threat to global public health. Novel antibiotics and treatment strategies are urgently needed. Murepavadin is a novel antibiotic that blocks the assembly of lipopolysaccharide (LPS) into the Pseudomonas aeruginosa outer membrane by inhibiting LPS transport protein D (LptD). Here, we demonstrated that murepavadin impairs bacterial outer membrane integrity, which induces the envelope stress response. We further found that the impaired outer membrane integrity increases the influx of β-lactam antibiotics, resulting in enhanced bactericidal effects. In addition, the combination of murepavadin and a β-lactam/β-lactamase inhibitor mixture (ceftazidime/avibactam) slowed down the resistance development of P. aeruginosa. Overall, this study demonstrates the bacterial response to murepavadin and provides a new combination strategy for effective treatment.

Ovarian Cancer-Insights into Platinum Resistance and Overcoming It.

Ovarian cancer is the most lethal gynecologic malignancy. Platinum-based chemotherapy is the backbone of treatment for ovarian cancer, and although the majority of patients initially have a platinum-sensitive disease, through multiple recurrences, they will acquire resistance. Platinum-resistant recurrent ovarian cancer has a poor prognosis and few treatment options with limited efficacy. Resistance to platinum compounds is a complex process involving multiple mechanisms pertaining not only to the tumoral cell but also to the tumoral microenvironment. In this review, we discuss the molecular mechanism involved in ovarian cancer cells' resistance to platinum-based chemotherapy, focusing on the alteration of drug influx and efflux pathways, DNA repair, the dysregulation of epigenetic modulation, and the involvement of the tumoral microenvironment in the acquisition of the platinum-resistant phenotype. Furthermore, we review promising alternative treatment approaches that may improve these patients' poor prognosis, discussing current strategies, novel combinations, and therapeutic agents.

CCT251545 enhances drug delivery and potentiates chemotherapy in multidrug-resistant cancers by Rac1-mediated macropinocytosis.

It was well known that P-glycoprotein (P-gp/ABCB1) is a master regulator of multidrug resistance (MDR) in cancers. However, the clinical benefit from blocking this pathway remains inconclusive, which motivates a paradigm shift towards alternative strategies for enhancing drug influx. Using a patient-derived organoid (PDO)-based drug screening platform, we report that the combined use of chemotherapy and CCT251545 (CCT) displays robust synergistic effect against PDOs and reduces proliferation of MDR cancer cells in vitro, and results in regression of xenograft tumors, reductions in metastatic dissemination and recurrence rate in vivo. The synergistic activity mediated by CCT can be mainly attributed to the intense uptake of chemotherapeutic agents into the cells, accompanied by alterations in cell phenotypes defined as a mesenchymal epithelial transformation (MET). Mechanistically, analysis of the transcriptome coupled with validation in cellular and animal models demonstrate that the chemosensitizing effect of CCT is profoundly affected by Rac1-dependent macropinocytosis. Furthermore, CCT binds to NAMPT directly, resulting in elevated NAD levels within MDR cancer cells. This effect promotes the assembly of adherents junction (AJ) components with cytoskeleton, which is required for continuous induction of macropinocytosis and consequent drug internalization. Overall, our results illustrate the potential use of CCT as a combination partner for the commonly used chemotherapeutic drugs in the management of MDR cancers.

Recent advances in the molecular basis of chemotherapy resistance and potential application of epigenetic therapeutics in chemorefractory renal cell carcinoma.

Among various types of cancers, kidney cancer is unique with respect to a low frequency of mutations and a relatively higher level of chemotherapy resistance. Resistance to chemotherapy is a major challenge in kidney cancer treatment in the clinic. Tremendous progress has been made in identifying the molecular changes associated with chemotherapy resistance in RCC. However, the exact contribution of these molecular changes to the acquisition of chemotherapy resistance is not fully understood. In addition to genetic changes, epigenetic alterations have been shown to contribute to various pathways associated with chemotherapy resistance, such as increased cell proliferation and survival, regulation of drug influx and efflux transporters, increased DNA repair, loss of DNA-damage-dependent apoptotic potential, cellular dedifferentiation to cancer stem cell, and epithelial-mesenchymal transitions (EMT). Moreover, recent studies suggest that epigenetic aberrations that can be reversed by epigenetic therapeutics can potentially be targeted to restore chemosensitivity in chemorefractory kidney cancer. This review article highlights current knowledge of the role of genetic and epigenetic aberrations as well as the physiological and metabolic changes associated with chemotherapeutic resistance. Additionally, current approaches and future directions for overcoming chemotherapeutic resistance including the potential of epigenetic therapeutic in chemorefractory kidney cancer have also been discussed. This article is categorized under: Cancer > Genetics/Genomics/Epigenetics.

Different effects of magnetic field on drug activity in human uterine sarcoma cell lines MES-SA and MES-SA/Dx5

ABSTRACT Previous studies reported that combined effect of magnetic field (MF) on cytotoxic drugs in human cancer cells. We focused on the effects of 60 Hz MF on drug activity in human uterine sarcoma MES-SA and drug-resistant variant MES-SA/Dx5 cells that overexpressed the membrane protein MDR1(P-glycoprotein), a drug efflux transporter for doxorubicin, daunorubicin, and etoposide, but not cisplatin. The cisplatin with MF caused 60% decrease in cell viability when compared with no MF treatment, cisplatin alone in MES-SA cells. Even in MES-SA/Dx5 cells, MF exposure equally enhanced cisplatin activity. Then, MF enhanced doxorubicin and daunorubicin activity in MES-SA cells and caused 60% decrease in the cell viability compared with these drugs only but had less effect on these drugs in MES-SA/Dx5 cells. Etoposide activity was unaffected by MF exposure in both cell lines, although etoposide is a MDR1 substrate as with doxorubicin and daunorubicin. Thus, MF had no direct impact on MDR1 in the cell membrane. However, the differences in doxorubicin and daunorubicin activity between MES-SA and MES-SA/Dx5 data revealed that the presence of MDR1 in abundance prevented the enhancing effects of MF on doxorubicin and daunorubicin activity. These results suggested that MF may act in the opposite direction of MDR1, affect the drug influx transporters for doxorubicin and daunorubicin, and facilitate anticancer drug uptake into the cells.

The Effect of Melatonin and Cisplatin Combination Using Copper-Transporting ATPase-1, P-Glycoprotein, and Gamma-Glutamylcysteinylglycine on Ovarian Cancer Biological Cell SKOV3

Background: Ovarian cancer is fifth most common female cancer and third most common cancer in Indonesia, but most are advanced stage patients that experiencing recurrence, which indicates resistance to treatment especially to cisplatin. Melatonin appears as an alternative that can support apoptotic effect of cisplatin as a chemotherapy regimen.
 Aim: To determine effect of combination melatonin and cisplatin compared with cisplatin only chemotherapy on chemotherapy resistance with Copper Transporting ATPase-1 (CTR-1), P-glycoprotein (P-Gp), and Gamma-Glutamylcysteinylglycine (GSH) biomarkers in ovarian cancer biological cells SKOV3
 Methods: This research design was experimental laboratory, post-test only control group design, using SKOV3 cell culture. This study was performed in the SCTE IMERI FKUI laboratory and Integrated Laboratory FKUI. MTS assay was used to calculate IC50 of each materials. The materials used were melatonin (concentration was 25,50,100,200,300 nM), cisplatin (concentration was 0.1, 0.5, 1, 2, 5 mM), and doxorubicin (concentration 10,20,40,50,80,100,200 µM). IC50 melatonin was 1,841 mM, IC50 cisplatin was 117,5 µM, and IC50 doxorubicin was 14,72 µM. Samples were control negative group, IC50 doxorubicin as a control positive, IC50 cisplatin, IC50 melatonin, combination group of melatonin and cisplatin were 1xIC50, ¾xIC50, ½xIC50, and ¼xIC50. ANOVA and Bonferroni test were used for statistical test.
 Results: Based on data processing, IC50 of melatonin was 1,841 mM, IC50 of doxorubicin was 14,72 mM, while IC50 of cisplatin was 117.5 μM. The mean expression of CTR-1 in IC50 melatonin group was 15.77 ± 0.21 and in IC50 cisplatin group was 10.87 ± 0.91, mean expression in IC50 doxorubicin group was 30,33 ± 0,4. Meanwhile, mean expression of CTR-1 in IC50 cisplatin was 7,37±0,7, and in combination 1 group (1xIC50 melatonin and 1xIC50 cisplatin) was 19,73±1.0,49. For P glycoprotein, mean expression in IC50 cisplatin was 16±1,59, in IC50 melatonin group was 7,37±0,21, in IC50 doxorubicin was 0, and in combination 1 group (1xIC50 melatonin and 1xIC50 cisplatin) was 6,7±0,17. Last, in GSH, mean expression in IC50 cisplatin group was 33,2±0,87, in IC50 melatonin group was 12,57±0,12, in IC50 doxorubicin group was 1,33±0,66, and in combination 1 group (1xIC50 melatonin and 1xIC50 cisplatin) was 11,73±0,67. There was significant difference of CTR-1 expression in combination 1 group which was higher (19.73%), p-glycoprotein expression in combination 1 group which was lower (6,7%), and also GSH expression in combination 1 group was lower (11,73%) compared to other groups.
 Conclusion: The group 1 combination of 1xIC50 melatonin and 1x IC50 cislatin with 1.841 mM and cisplatin 117.5 uM were able to reduce cisplatin chemotherapy resistance by increasing drug influx activity by increasing CTR-1 expression, decreasing drug efflux through decreasing p-glycoprotein expression, and decreased DNA repair activity through decreased GSH expression.

Single-treatment tumor ablation with photodynamic liposomal irinotecan sucrosulfate

Irinotecan (IRI) loaded actively into PEGylated liposomes via a sucrosulfate gradient has been approved recently to treat advanced pancreatic cancer. In this study, a similar liposomal composition was developed that includes a low mole fraction (1 mol.%) of porphyrin-phospholipid (PoP), a photosensitizer that stably incorporates into liposomes, to confer light-triggered IRI release. IRI-loaded PoP liposomes containing ammonium sucrosulfate (ASOS) as a complexing agent were more stable in serum compared to liposomes employing the more conventional ammonium sulfate. Without irradiation, PoP IRI liposomes released less than 5% IRI during 8 h of incubation in bovine serum at 37 °C, but released over 90% of the drug within minutes of exposure to red light (665 nm) irradiation. A single treatment with IRI-PoP liposomes and light exposure (15 mg/kg IRI with 250 J/cm2) resulted in tumor eradication in mice bearing either MIA PaCa-2 tumors or low-passage patient-derived tumor xenografts that recapitulate characteristics of the clinical disease. Analogous monotherapies of IRI or photodynamic therapy were ineffective in controlling tumor growth. Enhanced drug uptake could be visualized within laser-treated tumors by direct in situ imaging of irinotecan. Biodistribution analysis of IRI, its active metabolite (SN-38), and major metabolite (SN-38 G) showed that laser treatment significantly increased tumor accumulation of all IRI-derived molecular species. A pharmacokinetic model that hypothesized tumor vasculature permeabilization as the primary reason underlying the increased drug deposition accounted for the enhanced drug influx into tumors.

HopE and HopD Porin-Mediated Drug Influx Contributes to Intrinsic Antimicrobial Susceptibility and Inhibits Streptomycin Resistance Acquisition by Natural Transformation in Helicobacter pylori.

ABSTRACTHelicobacter pylori is a human pathogen competent for natural transformation. Intrinsic and acquired antibiotic resistance contribute to the survival and multiplication of H. pylori under antibiotics. While drug-resistance dissemination by natural transformation (NT)-mediated horizontal gene transfer remains poorly understood in H. pylori. The purpose of the study was to investigate the role of H. pylori porins (HopA, HopB, HopC, HopD, and HopE) in the intrinsic antibiotic resistance and to preliminarily reveal the potential effect of HopE and HopD porins in streptomycin resistance acquisition after NT in the presence of antibiotics. Using traditional antibiotic susceptibility tests and growth curve analysis, we found the MIC values of metronidazole, clarithromycin, levofloxacin, tetracycline, rifampin, and streptomycin in mutants lacking HopE and/or HopD were significantly elevated compare to those in wild-type strain. The quantitative analysis of the tetramethyl rhodamine isothiocyanate (TRITC)-labeled streptomycin accumulation at the single-cell level showed reduced streptomycin intracellular fluorescence in ΔhopE and ΔhopD mutant cells. Furthermore, in the presence of translation-inhibiting antibiotic streptomycin, the resistance acquisition frequency was decreased in the wild-type strain, which could be reversed by mutants lacking HopE and HopD that restored relatively high resistance acquisition frequencies. By transforming a pUC19-rpsLmut-sfgfp linear plasmid carrying a streptomycin conferring mutation, we observed that the impaired ability of rpsLmut synthesis in the wild-type strain was restored in the ΔhopE and ΔhopD mutant transformants. Our study revealed that in the presence of streptomycin, resistance acquisition at least partially relied on the deletion of the hopE and hopD genes, because their loss reduced streptomycin concentration in the cell and thus restored the expression of the resistance-conferring gene, which was inhibited by streptomycin in wild-type strain. The loss of HopE and HopD influx activity may also preserve resistance acquisition by transformation in the presence of antibiotics with other modes of action.IMPORTANCE Helicobacter pylori is constitutively competent for natural transformation (NT) and possesses an efficient system for homologous recombination, which could be utilized to study the NT-mediated horizontal gene transfer induced antibiotic resistance acquisition. Bacterial porins have drawn renewed attention because of their crucial role in antibiotic susceptibility. From the perspective of porin-mediated influx in H. pylori, our study preliminarily revealed the important role of HopE and HopD porins not only in preserving the intrinsic susceptibility to specific antibiotic but also in evading acquired antibiotic resistance by NT in the presence of translation-inhibiting antimicrobial. Therefore, the loss of HopE or HopD porin in H. pylori genomes, combined with the large number of secreted or cell-free genetic elements carrying mutations conferring antibiotic resistance, may raise the possibility that this mechanism plays a potential role in the propagation of antibiotic resistance within H. pylori communities.

Thiazole derivative based topical nanoemulgel for inhibition of bacterial virulence in surface infections.

Objective(s):Antimicrobial resistance emerged as a global challenge owing to limited therapeutic options to control infections. Pseudomonas aeruginosa, an MDR pathogen already developed resistance against many conventional antibiotics. An “anti-virulence strategy” that targets bacterial virulence rather than growth proves effective against drug-resistant pathogens. Materials and Methods:Here, we used a structure-based drug design approach to identify lead molecules using the LasR receptor protein of P. aeruginosa as a target responsible for virulence production in this bacterium. From the identified hits, we developed lead-based nanoformulation and investigated its effectiveness for treating the P. aeruginosa associated surface-infection in-vivo. First, TC-based nanoemulsions were fabricated by high-pressure homogenization and evaluated for various in vitro parameters. The optimized nanoemulsions were thereby utilized to prepare NEG.Results: The nanoemulsion (F3) exhibited low droplet size (51.04±1.88 nm), PDI (0.065±1.14), and negative zeta potential (-33.65±0.82 mV). In animals, topical application of NEG-3 demonstrated significant improvement on skin permeability (459±10.17 µg), drug influx (18.99±0.76 μg/cm2 hr), and repressed the CFU of P. aeruginosa induced-surface infection (P≤ 0.001). The histology of rat skin demonstrated a significant effect for groups treated with TC-based NEGs as compared with a negative control group, whereas no significant effect was seen on rat liver indicating low systemic exposure to the drug. Also, NEG3 showed no significant changes under different stability conditions after 3 months.Conclusion:TC-based NEGs open up the possibility of a more effective way to combat serious surface infections caused by P. aeruginosa.

Abstract P3-13-06: Single nucleotide polymorphisms and mortality after docetaxel-based chemotherapy in premenopausal breast cancer: A population-based cohort study in Denmark

Abstract Background Taxane-based chemotherapy is first line treatment in various cancers, including premenopausal breast cancer, but the inter-individual effectiveness is unpredictable. Differences in expression and activity of docetaxel-metabolizing enzymes and transporters (DMETs) may modify docetaxel effectiveness. Although reported data are inconsistent, some findings suggests that variant alleles that reduce the function of 1) SLC-transporters reduce drug influx into hepatocytes; 2) CYP-450 enzymes hamper drug metabolism; and 3) ABC-transporters decrease docetaxel clearance. The net effect is hypothesized to be increased docetaxel exposure and effectiveness. In contrast, GSTP1 variants have been associated with poorer docetaxel effectiveness. We investigated whether single nucleotide polymorphisms (SNPs) in drug metabolizing enzymes and transporters were associated with mortality in premenopausal breast cancer patients. Materials and methods Using the Danish Breast Cancer Group (DBCG) clinical database, we identified data on premenopausal women aged 18–55 years, diagnosed with non–metastatic breast cancer during 2007–2011. All women were recommended epirubicin, cyclophosphamide and docetaxel-based adjuvant chemotherapy, and tamoxifen if the disease was estrogen receptor (ER) positive. From DBCG and other Danish administrative and medical registries we retrieve data on death, emigration, and tumor characteristics. We collected archived formalin-fixed paraffin-embedded primary tumor tissue from Danish pathology departments. We genotyped 17 candidate SNPs using TaqMan SNP genotyping assays and, for each SNP, categorized the women as having two normal alleles (wildtype) or at least one variant allele. We followed the women from six months after breast cancer diagnosis until death, emigration or 30th June 2019, whichever came first. Comparing variant carriers with wildtype, we computed cumulative incidence proportions (CIPs) and used Poisson regression models to calculate unadjusted incidence rate ratios (IRRs) and associated 95% confidence intervals (CIs) of all–cause mortality. We stratified by ER status to evaluate interaction. Analyses were repeated for breast cancer specific mortality. Results Our cohort included 2,262 women. During follow–up (median 9.6 years, interquartile range: 8.4–11.0), 250 women died (CIP: 14%); 219 due to breast cancer (CIP: 11%). Genotyping was successful for ≥95% of the study cohort, with exception of two SNPs, which were excluded from analyses. We detected decreased mortality in variant carriers of SLCO1B1 rs2306283 (IRR: 0.75, 95% CI 0.58–0.97), and ABCB1 rs1128503 (IRR: 0.81, 95% CI: 0.63–1.05), ABCB1 rs2032582 (IRR: 0.80, 95% CI: 0.62–1.04) and ABCC2 rs12762549 (IRR: 0.80, 95% CI: 0.62–1.04). In contrast, mortality was increased in carriers of GSTP1 rs1138272 (IRR: 1.28, 95% CI: 0.93–1.76) and, unexpectedly, in carriers of CYP3A rs10273424 (IRR: 1.36, 95% CI: 0.99–1.86) variant carriers. Sensitivity analyses yielded similar findings. ER status did not modify the associations. Conclusions In this study, mortality may be associated with SNPs in ABCB1, ABCC2, SLCO1B1, CYP3A and GSTP1 in premenopausal women with non-metastatic breast cancer receiving docetaxel. The mechanisms underlying our findings remains unclear but may be related to docetaxel pharmacokinetics. Citation Format: Cathrine F Hjorth, Per Damkier, Tore B Stage, Søren Feddersen, Bent Ejlertsen, Timothy L Lash, Stephen Hamilton-Dutoit, Thomas P Ahern, Mikael Rørth, Henrik T Sørensen, Deirdre Cronin-Fenton. Single nucleotide polymorphisms and mortality after docetaxel-based chemotherapy in premenopausal breast cancer: A population-based cohort study in Denmark [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-13-06.

Non-ionic Surfactants as a P-Glycoprotein(P-gp) Efflux Inhibitor for Optimal Drug Delivery-A Concise Outlook.

Significant research efforts have been devoted to unraveling the mystery of P-glycoprotein(P-gp) in drug delivery applications. The efflux membrane transporter P-gp is widely distributed in the body and accountable for restricting drug absorption and bioavailability. For these reasons, it is the primary cause of developing multidrug resistance (MDR) in most drug delivery applications. Therefore, P-gp inhibitors must be explored to address MDR and the low bioavailability of therapeutic substrates. Several experimental models in kinetics and dynamic studies identified the sensitivity of drug molecules and excipients as a P-gp inhibitor. In this review, we aimed to emphasize nonionic surface-active agents for effective reversal of P-gp inhibition. As it is inert, non-toxic, noncharged, and quickly reaching the cytosolic lipid membrane (the point of contact with P-gp efflux protein) enables it to be more efficient as P-gp inhibitors. Moreover, nonionic surfactant improves drug absorption and bioavailability through the various mechanism, involving (i) association of drug with surfactant improves solubilization, facilitating its cell penetration and absorption; (ii) weakening the lateral membrane packing density, facilitating the passive drug influx; and (iii) inhibition of the ATP binding cassette of transporter P-glycoprotein. The application of nonionic surfactant as P-gp inhibitors is well established and supported by various experiments. Altogether, herein, we have primarily focused on various nonionic surfactants and their development strategies to conquer the MDR-causing effects of P-gp efflux protein in drug delivery. Graphical Abstract.

MiR-197-5p increases Doxorubicin-mediated anticancer cytotoxicity of HT1080 fibrosarcoma cells by decreasing drug efflux

Doxorubicin (Dox) is one of the most used drugs in the treatment of Soft tissue sarcoma. However, acquired resistance linked with poor survival and numerous side effects are the major challenges. Meanwhile, miRNAs are reported to influence the chemotherapeutic responses. However, there is hardly any evidence on the involvement of tumor-suppressive miR-197 reported in our previous study in augmenting the sensitivity of fibrosarcoma cells to Dox. Therefore, in this study, we intend to decipher if miR-197–5p combined with Dox could increase the anticancer cytotoxicity. For this, we evaluated the antitumorigenic effects of Dox and miR-197–5p individually and in combination by performing a series of molecular assays. We noticed that the sub-lethal concentration of miR-197–5p markedly enhanced the sensitivity of HT1080 fibrosarcoma cells to Dox by promoting apoptosis and G2/M cell cycle arrest. We also observed miR-197–5p sensitizes HT1080 cells to Dox by increasing drug influx, possibly due to suppression of MDR genes (ABCC1, MVP). Moreover, we found that KIAA0101, a target of miR-197–5p is inhibited by Dox, which is further repressed when treated in combination with miRNA. We also observed a marked upregulation of p53, known to be negatively correlated with KIAA0101 in Dox and miR-197–5p combination treatment compared to Dox alone. Taken together, our study revealed that Dox chemotherapy in combination with miR-197–5p could overcome the problem of drug efflux and enhance its antitumor effects on fibrosarcoma.

Quinolone Resistance of Actinobacillus pleuropneumoniae Revealed through Genome and Transcriptome Analyses.

Actinobacillus pleuropneumoniae is a pathogen that infects pigs and poses a serious threat to the pig industry. The emergence of quinolone-resistant strains of A. pleuropneumoniae further limits the choice of treatment. However, the mechanisms behind quinolone resistance in A. pleuropneumoniae remain unclear. The genomes of a ciprofloxacin-resistant strain, A. pleuropneumoniae SC1810 and its isogenic drug-sensitive counterpart were sequenced and analyzed using various bioinformatics tools, revealing 559 differentially expressed genes. The biological membrane, plasmid-mediated quinolone resistance genes and quinolone resistance-determining region were detected. Upregulated expression of efflux pump genes led to ciprofloxacin resistance. The expression of two porins, OmpP2B and LamB, was significantly downregulated in the mutant. Three nonsynonymous mutations in the mutant strain disrupted the water–metal ion bridge, subsequently reducing the affinity of the quinolone–enzyme complex for metal ions and leading to cross-resistance to multiple quinolones. The mechanism of quinolone resistance in A. pleuropneumoniae may involve inhibition of expression of the outer membrane protein genes ompP2B and lamB to decrease drug influx, overexpression of AcrB in the efflux pump to enhance its drug-pumping ability, and mutation in the quinolone resistance-determining region to weaken the binding of the remaining drugs. These findings will provide new potential targets for treatment.

Overcoming anti-cancer drug resistance via restoration of tumor suppressor gene function

The cytotoxic anti-cancer drugs cisplatin, paclitaxel, doxorubicin, 5-fluorouracil (5-FU), as well as targeted drugs including imatinib, erlotinib, and nivolumab, play key roles in clinical cancer treatment. However, the frequent emergence of drug resistance severely comprosises their anti-cancer efficacy. A number of studies indicated that loss of function of tumor suppressor genes (TSGs) is involved in the development of cancer drug resistance, apart from decreased drug influx, increased drug efflux, induction of anti-apoptosis mechanisms, alterations in tumor microenvironment, drug compartmentalization, enhanced DNA repair and drug inactivation. TSGs are involved in the pathogenesis of tumor formation through regulation of DNA damage repair, cell apoptosis, autophagy, proliferation, cell cycle progression, and signal transduction. Our increased understanding of TSGs in the past decades demonstrates that gene mutation is not the only reason that leads to the inactivation of TSGs. Loss of function of TSGs may be based on the ubiquitin-proteasome pathway, epigenetic and transcriptional regualtion, post-translation modifications like phosphorylation as well as cellular translocation of TSGs. As the above processes can constitute"druggable targets", these mechanisms provide novel therapeutic approaches in targeting TSGs. Some small molecule compounds targeting these approaches re-activated TSGs and reversed cancer drug resistance. Along this vein, functional restoration of TSGs is a novel and promising approach to surmount cancer drug resistance. In the current review, we draw a scenario based on the role of loss of function of TSGs in drug resistance, on mechanisms leading to inactivation of TSGs and on pharmacological agents acting on these mechanisms to overcome cancer drug resistance. This review discusses novel therapeutic strategies targeting TSGs and offers possible modalities to conquer cancer drug resistance.

Association between Gene Expression Levels of Candidate Cytarabine Pathway and Treatment Outcome Using Publically Available Data from Target Project

Acute myeloid leukemia (AML) is the second most common form of childhood leukemia and has the worst prognosis of all major childhood cancers. Improving the treatment outcome for patients with AML remains a major clinical challenge. The nucleoside analog, cytarabine (Ara-C), has been the mainstay of AML chemotherapy for more than 40 years. However, extensive inter-patient variation in treatment response, development of resistance, and severe toxicity remain as major hurdles to effective Ara-C chemotherapy. It is a pro-drug that requires activation to Ara-CTP by multiple phosphorylation steps. Thus, inter-patient variation in gene expression levels of candidate genes involved in cytarabine metabolism can impact therapeutic outcome. In last few years' significant efforts have been invested in generating genomic, transcriptomic and epigenetic data in various cancer patients including AML. TCGA (The Cancer Genome Atlas) is one of the most commonly available such resource, recently another resource available to scientific community TARGET (Therapeutically Applicable Research to Generate Effective Treatments), a project initiated by the National Cancer Institute (NCI) in 2006. This databased was created with the aim to take multi-omics integrative approach to characterize molecular alterations associated with poor outcomes in some common children's cancers including AML. In this study, we leveraged TARGET database to extract gene expression levels (generated using Affymetrix Gene ST Array) to investigate whether inter-patient differences in expression levels of 11 key candidate genes in cytarabine metabolic pathway are predictive of minimal residual disease (MRD: positive or negative) and thus outcome. These genes included DCK and CMPK1 encoding for drug activating enzymes, CDA, DCTD, NT5C2, NT5C3A, important for cytarabine inactivation; drug influx transporter SLC29A1; RRM1 and RRM2 as ribonucleotide inhibitors; CTPS1 and NME3 which play a key role in the synthesis of nucleoside tri-phosphates. We also investigated correlation of the expression levels of these genes with each other to get insight into whether these genes could be co-regulated. In order to have consistency we only included data (gene expression and clinical and patient demographic, characteristics) from 105 AML patients where expression levels were available from bone marrow specimens and also restricted our analysis to AAML03P1 and AAML0531 clinical cohorts. Our resultsshow significant correlation in expression levels between cytarabine metabolic pathway genes resulting in three clusters: First cluster included DCK, NT5C2, NT5C3A, RRM1 and CMPK1 with positive correlation with each other's ranging between (r=0.619 to 0.720 p-value Our results from association analysis with MRD showed that cluster 1 genes had higher expression levels in patients with positive MRD. On the other hand, cluster 2 genes have all a higher expression in the MRD negative. To get the combinatorial effect of gene-expression on different clinical endpoints we defined a combination score (CS) using CS = (DCK*NT5C2*NT5C3A*CMPK1*CTPS1*RRM1*RRM2*DCTD)/(CDA*NME3*SLC29A1). Higher CS was associated with positive MRD (P= 0.012) (Figure 2) and lower overall survival rates (r= - 0.232, P-value = 0.017). By generating a combinatorial expression score for cytarabine pathway genes, we have been able to identify features that might predict MRD, overall survival and thus outcome in patients treated with cytarabine. An interesting fact that we observed positive correlations between activating genes - DCK, CMPK with inactivating genes such as NT5C2 or reductases RRM1/RRM2 that negatively impact the drug activation. One possible explanation, when DCK is highly expressed, CMPK1 responsible for the second phosphorylation step increases. At the same time, RRM1 and RRM2 known as DCK inhibitors and NT5C2 become highly expressed to balance high DCK level. This might also be a plausible reason for not observing consistent correlations between expression levels of ara-C pathway genes with outcome. Download : Download high-res image (178KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

Dual-Function Potentiation by PEG-BPEI Restores Activity of Carbapenems and Penicillins against Carbapenem-Resistant Enterobacteriaceae.

The rise of life-threatening carbapenem-resistant Enterobacteriaceae (CRE) infections has become a critical medical threat. Some of the most dangerous CRE bacteria can produce enzymes that degrade a wide range of antibiotics, including carbapenems and β-lactams. Infections by CRE have a high mortality rate, and survivors can have severe morbidity from treatment with toxic last-resort antibiotics. CRE have mobile genetic elements that transfer resistance genes to other species. These bacteria also circulate throughout the healthcare system. The mobility and spread of CRE need to be curtailed, but these goals are impeded by having few agents that target a limited range of pathogenic CRE species. Against CRE possessing the metallo-β-lactamase NDM-1, Klebsiella pneumoniae ATCC BAA-2146 and Escherichia coli ATCC BAA-2452, the potentiation of meropenem and imipenem is possible with low-molecular weight branched polyethylenimine (600 Da BPEI) and its poly(ethylene glycol) (PEG)ylated derivative (PEG-BPEI) that has a low in vivo toxicity. The mechanism of action is elucidated with fluorescence assays of drug influx and isothermal calorimetry data showing the chelation of essential Zn2+ ions. These results suggested that 600 Da BPEI and PEG-BPEI may also improve the uptake of antibiotics and β-lactamase inhibitors. Indeed, the CRE E. coli strain is rendered susceptible to the combination of piperacillin and tazobactam. These results expand the possible utility of 600 Da BPEI potentiators, where previously we have demonstrated the ability to improve antibiotic efficacy against antibiotic resistant clinical isolates of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis.