Cisplatin Uptake Research Articles

CISPLATIN UPTAKE IN PERITONEAL METASTASES OF OVARIAN CANCER FOLLOWING CRS+HIPEC

Background: While the role of cisplatin HIPEC added to cytoreductive surgery in stage III epithelial ovarian cancer in improving outcomes becomes more clear, in vivo data on pharmacokinetics and -dynamics are lacking. Furthermore, factors such as the impact of hyperthermia remain to be defined. Here, we report in vivo tumor uptake of cisplatin of patient enrolled in the OvIP study (NCT02567253), a multicenter randomized trial investigating tumor tissue platinum penetration after normothermic (37°C) versus hyperthermic (41°C) chemoperfusion with cisplatin at 75 or 120 mg/m2.

Role of Organic Cation Transporter 2 in Autophagy Induced by Platinum Derivatives.

The human organic cation transporter 2 (hOCT2) mediates renal and neuronal cellular cisplatin and oxaliplatin uptake, and therefore plays a significant role in the development of side effects associated with these chemotherapeutic drugs. Autophagy is induced by cisplatin and oxaliplatin treatment and is believed to promote cell survival under stressful conditions. We examined in vitro the role of hOCT2 on autophagy induced by cisplatin and oxaliplatin. We also explored the effect of autophagy on toxicities of these platinum derivatives. Our results indicate that autophagy, measured as LC3 II accumulation and reduction in p62 expression level, is induced in response to cisplatin and oxaliplatin in HEK293-hOCT2 but not in wild-type HEK293 cells. Furthermore, inhibition of autophagy is associated with higher toxicity of platinum derivatives, and starvation was found to offer protection against cisplatin-associated toxicity. In conclusion, activation of autophagy could be a potential strategy to protect against unwanted toxicities induced by treatment with platinum derivatives.

Oocyte CTR1 is not essential for cisplatin-induced oocyte death of primordial follicle.

Accumulated evidence indicates that cisplatin, a platinum-based alkylating agent, causes preferential DNA damage to oocytes of primordial follicles (PFs) in the ovary, suggesting oocyte-favored accumulation of cisplatin. Copper transporter 1 (CTR1; Slc31a1 ) is implicated in facilitating cisplatin uptake in cells. Here we found that oocytes of PFs had constitutively higher expression of CTR1 than other cell types in mouse ovary. However, oocyte-specific Slc31a1 knockout was not sufficient to prevent cisplatin-induced depletion of PFs in vitro . Our data indicate that CTR1 would not be the only route for cisplatin to be transported inside the oocytes of PFs in the ovary.

Cisplatin Uptake in Macrophage Subtypes at the Single-Cell Level by LA-ICP-TOFMS Imaging.

A high-throughput laser ablation–inductively coupled plasma–time-of-flight mass spectrometry (LA-ICP-TOFMS) workflow was implemented for quantitative single-cell analysis following cytospin preparation of cells. For the first time, in vitro studies on cisplatin exposure addressed human monocytes and monocyte-derived macrophages (undifferentiated THP-1 monocytic cells, differentiated M0 macrophages, as well as further polarized M1 and M2 phenotypes) at the single-cell level. The models are of particular interest as macrophages comprise the biggest part of immune cells present in the tumor microenvironment and play an important role in modulating tumor growth and progression. The introduced bioimaging workflow proved to be universally applicable to adherent and suspension cell cultures and fit-for-purpose for the quantitative analysis of several hundreds of cells within minutes. Both, cross-validation of the method with single-cell analysis in suspension for THP-1 cells and with LA-ICP-TOFMS analysis of adherent M0 cells grown on chambered glass coverslips, revealed agreeing platinum concentrations at the single-cell level. A high incorporation of cisplatin was observed in M2 macrophages compared to the M0 and M1 macrophage subtypes and the monocyte model, THP-1. The combination with bright-field images and monitoring of highly abundant endogenous elements such as phosphorus and sodium at a high spatial resolution allowed assessing cell size and important morphological cell parameters and thus straightforward control over several cell conditions. This way, apoptotic cells and cell debris as well as doublets or cell clusters could be easily excluded prior to data evaluation without additional staining.

Oxidative Stress and Inflammation Caused by Cisplatin Ototoxicity.

Hearing loss is a significant health problem that can result from a variety of exogenous insults that generate oxidative stress and inflammation. This can produce cellular damage and impairment of hearing. Radiation damage, ageing, damage produced by cochlear implantation, acoustic trauma and ototoxic drug exposure can all generate reactive oxygen species in the inner ear with loss of sensory cells and hearing loss. Cisplatin ototoxicity is one of the major causes of hearing loss in children and adults. This review will address cisplatin ototoxicity. It includes discussion of the mechanisms associated with cisplatin-induced hearing loss including uptake pathways for cisplatin entry, oxidative stress due to overpowering antioxidant defense mechanisms, and the recently described toxic pathways that are activated by cisplatin, including necroptosis and ferroptosis. The cochlea contains G-protein coupled receptors that can be activated to provide protection. These include adenosine A1 receptors, cannabinoid 2 receptors (CB2) and the Sphingosine 1-Phosphate Receptor 2 (S1PR2). A variety of heat shock proteins (HSPs) can be up-regulated in the cochlea. The use of exosomes offers a novel method of delivery of HSPs to provide protection. A reversible MET channel blocker that can be administered orally may block cisplatin uptake into the cochlear cells. Several protective agents in preclinical studies have been shown to not interfere with cisplatin efficacy. Statins have shown efficacy in reducing cisplatin ototoxicity without compromising patient response to treatment. Additional clinical trials could provide exciting findings in the prevention of cisplatin ototoxicity.

The reversal of drug resistance by two-dimensional titanium carbide Ti2 C (2D Ti2C) in non-small-cell lung cancer via the depletion of intracellular antioxidant reserves.

BackgroundChemoresistance is a major barrier limiting the therapeutic efficacy of late stage non‐small cell lung cancer (NSCLC). In this study, we sought to use two‐dimensional titanium carbide (2D Ti2C) to reverse cisplatin resistance in NSCLC.MethodsWe first achieved favorable properties as a potential anti‐tumor agent. We then compared cell viability and cisplatin uptake in chemoresistant NSCLC cells before and after the use of 2D Ti2C. Afterwards, we explored the effects of 2D Ti2C on intracellular antioxidant reserves, followed by evaluating the subsequent changes in the expression of core drug resistance genes. Finally, we confirmed the tumor inhibitory effect and bio‐safety of 2D Ti2C in a drug‐resistant lung cancer model in nude mice.ResultsDue to the properties of thin layer, large specific surface area, and abundant reactive groups on the surface, 2D Ti2C can deplete the antioxidant reserve systems such as the glutathione redox buffer system, γ‐glutamylcysteine synthetase (γ‐GCS), glutathione peroxidase (GPx), glutathione‐S‐transferase‐Pi (GST‐π), and metallothionein (MT), thereby increasing the intracellular accumulation of cisplatin and decreasing the expression of drug resistance genes.Conclusions2D Ti2C can reverse NSCLC chemoresistance both in vitro and in vivo, suggesting that it may potentially become a novel and effective means to treat chemoresistant NSCLC in the clinic.

Cisplatin uptake and release assessment from hydrogel synthesized in acidic and neutral medium: An experimental and molecular dynamics simulation study

Targeted drug delivery systems (TDDS) are currently considered to overcome traditional drug delivery systems’ criticisms and weaknesses. Biodegradable hydrogels are widely used for the design of appropriate TDDS. Among them, the gum-based hydrogel is known as a proper material to produce drug delivery systems. The present research aimed to design a biodegradable hydrogel with maximum swelling rate, optimum drug loading, and efficient release rate. The acquired experimental findings have been compared to the molecular dynamics (MD) simulation results. Therefore, a response surface methodology (D-optimal method) was used to optimize the guar gum-based hydrogel’s physicochemical properties. The D-optimal method was employed to design an experiment (DOE) to evaluate significant variables, including initiator concentration, monomer concentration, cross-linker, pH, reaction temperature, and reaction time. Findings showed the optimum swelling rate was 4903.876%. Then, the loading and release of cisplatin, selected as the hydrogel structure model drug, were investigated using the molecular dynamics simulation technique. Cisplatin’s uptake and release from the hydrogel structure were higher in an acidic environment than in a neutral medium.

Wedelolactone protects against cisplatin-induced nephrotoxicity in mice via inhibition of organic cation transporter 2.

The balance of cisplatin uptake and efflux, mediated mainly by organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1), respectively, determines the renal accumulation and nephrotoxicity of cisplatin. Using transporter-mediated cellular uptake assay, we identified wedelolactone (WEL), a medicinal plant-derived natural compound, is a competitive inhibitor of OCT2 and a noncompetitive inhibitor of MATE1. Wedelolactone showed a selectivity to inhibit OCT2 rather than MATE1. Cytotoxicity studies revealed that wedelolactone alleviated cisplatin-induced cytotoxicity in OCT2-overexpressing HEK293 cells, whereas it did not alter the cytotoxicity of cisplatin in various cancer cell lines. Additionally, wedelolactone altered cisplatin pharmacokinetics, reduced kidney accumulation of cisplatin, and ameliorated cisplatin-induced acute kidney injury in the Institute of Cancer Research mice. In conclusion, these findings suggest a translational potential of WEL as a natural therapy for preventing cisplatin-induced nephrotoxicity and highlight the need for drug-drug interaction investigations of WEL with other treatments which are substrates of OCT2 and/or MATE1.

Preferential Cochleotoxicity of Cisplatin.

Cisplatin-induced ototoxicity in humans is more predominant in the cochlea than in the vestibule. Neither definite nor substantial vestibular dysfunction after cisplatin treatment has been consistently reported in the current literature. Inner ear hair cells seem to have intrinsic characteristics that make them susceptible to direct exposure to cisplatin. The existing literature suggests, however, that cisplatin might have different patterns of drug trafficking across the blood-labyrinth-barrier, or different degrees of cisplatin uptake to the hair cells in the cochlear and vestibular compartments. This review proposes an explanation for the preferential cochleotoxicity of cisplatin based on current evidence as well as the anatomy and physiology of the inner ear. The endocochlear potential, generated by the stria vascularis, acting as the driving force for hair cell mechanoelectrical transduction might also augment cisplatin entry into cochlear hair cells. Better understanding of the stria vascularis might shed new light on cochleotoxic mechanisms and inform the development of otoprotective interventions to moderate cisplatin associated ototoxicity.

Low intensity ultrasound enhances cisplatin uptake in vitro by cochlear hair cells.

Drug delivery to the inner ear has been challenging due to the blood-labyrinth barrier. Intracochlear drug delivery is an invasive alternative with less pharmacokinetic variables. In this study, the effect of low intensity ultrasound on drug uptake by hair cells is investigated. Cochlear explants harvested from newborn mice were cultured in a medium containing cisplatin to emulate drug delivered to the endolymph. The results demonstrated the exposure to ultrasound stimulation effectively enhanced cisplatin uptake by hair cells. The uptake started from the apical side of the hair cells and progressed inward as the exposure time increased.

Cisplatin uptake and release in pH sensitive zeolitic imidazole frameworks

Cancer remains hard to treat, partially due to the non-specificity of chemotherapeutics. Metal-organic frameworks (MOFs) are promising carriers for targeted chemotherapy, yet, to date, there have been few detailed studies to systematically enhance drug loading while maintaining controlled release. In this work, we investigate which molecular simulation methods best capture the experimental uptake and release of cisplatin from UiO-66 and UiO-66(NH2). We then screen a series of biocompatible, pH-sensitive zeolitic imidazolate frameworks (ZIFs) for their ability to retain cisplatin in healthy parts of the patient and release it in the vicinity of a tumor. Pure-component GCMC simulations show that the maximum cisplatin loading depends on the pore volume. To achieve this maximum loading in the presence of water, either the pore size needs to be large enough to occupy both cisplatin and its solvation shell or the MOF-cisplatin interaction must be more favorable than the cisplatin-shell interaction. Both solvated and non-solvated simulations show that cisplatin release rates can be controlled by either decreasing the pore limiting diameters or by manipulating framework-cisplatin interaction energies to create strong, dispersed adsorption sites. The latter method is preferable if cisplatin loading is performed from solution into a pre-synthesized framework as weak interaction energies and small pore window diameters will hinder cisplatin uptake. Here, ZIF-82 is most promising. If it is possible to load cisplatin during crystallization, ZIF-11 would outcompete the other MOFs screened as cisplatin cannot pass through its pore windows; therefore, release rates would be purely driven by the pH triggered framework degradation.

Regulation of Anion Channel LRRC8 Volume-Regulated Anion Channels in Transport of 2'3'-Cyclic GMP-AMP and Cisplatin under Steady State and Inflammation.

The recently identified anion channel LRRC8 volume-regulated anion channels (VRACs) are heteromeric hexamers constituted with the obligate LRRC8A subunit paired with at least one of the accessory LRRC8B to LRRC8E subunits. In addition to transport chloride, taurine, and glutamate, LRRC8 VRACs also transport the anticancer agent cisplatin and STING agonists 2'3'-cyclic GMP-AMP (cGAMP) and cyclic dinucleotides; hence, they are implicated in a variety of physiological and pathological processes, such as cell swelling, stroke, cancer, and viral infection. Although the subunit composition largely determines VRAC substrate specificity, the opening of various VRAC pores under physiological and pathological settings remains enigmatic. In this study, we demonstrated that VRACs comprising LRRC8A and LRRC8E (LRRC8A/E-containing VRACs), specialized in cGAMP transport, can be opened by a protein component present in serum under resting condition. Serum depletion ablated the tonic activity of LRRC8A/E-containing VRACs, decreasing cGAMP transport in various human and murine cells. Also, heating or proteinase K treatment abolished the ability of serum to activate VRAC. Genetic analyses revealed a crucial role for cGAMP synthase (cGAS) in serum/TNF-promoted VRAC activation. Notably, the presence of cGAS on the plasma membrane, rather than its DNA-binding or enzymatic activity, enabled VRAC activation. Moreover, phospholipid PIP2 seemed to be instrumental in the membrane localization of cGAS and its association with VRACs. Corroborating a role for LRRC8A/D-containing VRACs in cisplatin transport, serum and TNF markedly potentiated cisplatin uptake and killing of cancer cells derived from human or mouse. Together, these observations provide new insights into the complex regulation of VRAC activation and suggest a novel approach to enhance the efficacy of cGAMP and cisplatin in treating infection and cancer.

In Situ Supramolecular Self‐Assembly of Pt(IV) Prodrug to Conquer Cisplatin Resistance

AbstractDrug resistance has always been a huge challenge that should be urgently conquered to improve the efficacy of anticancer drugs. Herein, a synergistic Pt(IV) prodrug, Npx‐pp‐Pt(IV), is proposed, combining dual responsive behavior with dual drug resistance‐related pathways deactivation. First, Npx‐pp‐Pt(IV) can in situ form a supramolecular self‐assembly with a nanofiber structure on the cancer cell surface triggered by phosphatase, which confines the drug in the tumor and effectively enhances the cellular uptake of cisplatin, resulting in a high cancer cell selectivity and an extremely low non‐targeted cytotoxicity. After being endocytosed, the self‐assembly shows glutathione‐responsive cisplatin release and reverses the IC50 of cisplatin‐resistant cancer cells to that of sensitive ones. Second, the obtained Pt(IV) prodrug can significantly damage cisplatin‐resistance cancer cells through cyclooxygenase‐2 and nuclear factor‐kappa B‐mediated apoptosis pathways, which benefit from the integration of naproxen into the prodrug. The in vivo experiment demonstrates a tumor inhibition rate of 80%. Therefore, Npx‐pp‐Pt(IV) is a multispecific cisplatin derivative, and in situ self‐assembly is believed to be a new strategy to conquer drug resistance for clinical care.

Use of C. elegans as a 3R-compliant in vivo model for the chemoprevention of cisplatin-induced neurotoxicity

Anticancer therapeutics can provoke severe side effects that impair the patient's quality of life. A frequent dose-limiting side effect of platinum-based anticancer therapy is neurotoxicity. Its pathophysiology is poorly understood, and effective preventive or therapeutic measures are missing. Therefore, elucidation of the molecular mechanism of platinating drug-induced neurotoxicity and the development of preventive strategies is urgently needed. To this end, we aim to use C. elegans as a 3R-compliant in vivo model. The 3R principles were conceived for animal welfare in science concerning animal experiments, which should be replaced, reduced or refined. We can analytically demonstrate dose-dependent uptake of cisplatin (CisPt) in C. elegans, as well as genotoxic and cytotoxic effects based on DNA adduct formation (i.e., 1,2-GpG intrastrand crosslinks), induction of apoptosis, and developmental toxicity. Measuring the impairment of pharyngeal pumping as a marker of neurotoxicity, we found that especially CisPt reduces the pumping frequency at concentrations where basal and touch-provoked movement were not yet affected. CisPt causes glutathione (GSH) depletion and RNAi-mediated knockdown of the glutamate-cysteine ligase GCS-1 aggravates the CisPt-induced inhibition of pharyngeal pumping. Moreover, N-acetylcysteine (NAC) mitigated CisPt-triggered toxicity, indicating that GSH depletion contributes to the CisPt-induced pharyngeal damage. In addition to NAC, amifostine (WR1065) also protected the pharynx of C. elegans from the toxic effects of CisPt. Measuring pharyngeal activity by the electrophysiological recording of neurotransmission in the pharynx, we confirmed that CisPt is neurotoxic in C. elegans and that NAC is neuroprotective in the nematode. The data support the hypothesis that monitoring the pharyngeal activity of C. elegans is a useful surrogate marker of CisPt-induced neurotoxicity. In addition, a low GSH pool reduces the resistance of neurons to CisPt treatment, and both NAC and WR1065 are capable of attenuating platinum-induced neurotoxicity during post-incubation in C. elegans. Overall, we propose C. elegans as a 3R-compliant in vivo model to study the molecular mechanisms of platinum-induced neurotoxicity and to explore novel neuroprotective therapeutic strategies to alleviate respective side effects of platinum-based cancer therapy.

Ultrasmall iron oxide nanoparticles cisplatin (IV) prodrug nanoconjugate: ICP-MS based strategies to evaluate the formation and drug delivery capabilities in single cells

Ultrasmall iron oxide nanoparticles (<10 nm) were explored here as nanotransporters of cis-diamminetetrachloroplatinum (IV) (a cisplatin prodrug) in cellular models. The coating of the particles containing reactive carboxylic acid groups enabled the formation of a stable conjugate between the prodrug and the nanoparticles using one pot reaction. The nanoconjugate was characterized by different techniques exhibiting diameters of about 6.6 ± 1.0 nm. The use of a hyphenated strategy based on high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) permitted the quantitative evaluation of Fe and Pt in the nanoconjugate. Furthermore, the cellular uptake of the synthetic nanoconjugate was explored by single cell-ICP-MS (SC-ICP) which was used for the first time in this type of studies. The experiments in A2780 and A2780cis, sensitive and resistant ovarian cancer cell models respectively, revealed intracellular platinum concentrations of 12 fg/cell and 4 fg/cell, respectively which were 4-fold higher with respect to the uptake of cisplatin in both models. Intracellular drug release from the nanoconjugate was proved by measuring DNA platination in the same cells. In this case, levels of about 250 ng Pt/mg DNA were observed, about 5-fold higher when the nanoconjugate was used in comparison to cisplatin. Furthermore, the differences between the two lines turned to be significantly smaller than in the case of using cisplatin. The quantitative analytical tools developed here provided essential information required to fully characterize the developed nanoplatforms particularly important to overcome drug resistance.

Dual-mode imaging of copper transporter 1 in HepG2 cells by hyphenating confocal laser scanning microscopy with laser ablation ICPMS.

Copper transporter 1 (CTR1) is a transport protein involved in copper and cisplatin uptake. The visualization of cellular CTR1 migration and its redistribution is highly important in copper/cisplatin exposure/transport. However, to the best of our knowledge, this is a highly challenging task. Herein, a dual-mode imaging strategy for CTR1 is developed by hyphenating confocal laser scanning microscopy (CLSM) and laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) with a fluorescent/elemental bifunctional tag conjugated with anti-CTR1 antibody. The tag consists of rhodamine B and zirconium metal-organic frameworks (Zr-MOF) for CLSM fluorescence imaging and LA-ICPMS element imaging for a same group of HepG2 cells in a designated visual zone. This dual-mode imaging strategy facilitates visualization of CTR1 migration and meanwhile provides information of CTR1 redistribution in HepG2 cells by uptake of divalent copper or cisplatin. The present dual-mode imaging strategy provides in-depth information for the elucidation of CTR1 involved biological processes. Graphical abstract.

Palladium and platinum complexes of folic acid as new drug delivery systems for treatment of breast cancer cells

Cisplatin is administrated as an agent in treatment of various cancers by intercalation between DNA strands and inhibition of DNA replication. Among the various factors, two important reasons like inhibition of apoptosis by anti-apoptotic mechanisms and inability to distinguish normal cells from cancerous led us to synthesize two new complexes of platinum and palladium containing folic acid (FA). Following, the cytotoxic effects of complexes were studied using MTT assay in MCF-7 cells, then expression levels of Bak1, Bclx and Caspase-3 genes were elucidated by Real-time RT-PCR technique. Also, the behavior of complexes in the binding sites of folate receptor was described by molecular docking. All experiments have confirmed that presence of FA could improve cytotoxicity, apoptosis and cellular uptake of cisplatin in MCF-7 breast cancer cells. In addition, formation of 2,2′-bipyridine palladium(II) chelate with FA decreased the anti-apoptotic Bclx levels in comparison with cisplatin.

Iron sucrose ('RBT-3') activates the hepatic and renal HAMP1 gene, evoking renal hepcidin loading and resistance to cisplatin nephrotoxicity.

Iron sucrose (FeS) administration induces a state of renal preconditioning, protecting against selected forms of acute kidney injury (AKI). Recent evidence suggests that recombinant hepcidin also mitigates acute renal damage. Hence the goals of this study were to determine whether a new proprietary FeS formulation ('RBT-3') can acutely activate the hepcidin (HAMP1) gene in humans, raising plasma and renal hepcidin concentrations; assess whether the kidney participates in this posited RBT-3-hepcidin generation response; test whether RBT-3 can mitigate a clinically relevant AKI model (experimental cisplatin toxicity) and explore whether mechanisms in addition to hepcidin generation are operative in RBT-3's cytoprotective effects. Healthy human volunteers (n = 9) and subjects with Stages 3-4 CKD (n = 9) received 120, 240 or 360 mg of RBT-3 (intravenously over 2 h). Plasma and urine samples were collected and assayed for hepcidin levels (0-72 h post-RBT-3 injection). In complementary mouse experiments, RBT-3 effects on hepatic versus renal hepcidin (HAMP1) messenger RNA (mRNA) and protein levels were compared. RBT-3's impact on the mouse Nrf2 pathway and on experimental cisplatin nephrotoxicity was assessed. Direct effects of exogenous hepcidin on in vivo and in vitro (HK-2 cells) cisplatin toxicity were also tested. RBT-3 induced rapid, dose-dependent and comparable plasma hepcidin increases in both healthy volunteers and chronic kidney disease subjects (∼15 times baseline within 24 h). Human kidney hepcidin exposure was confirmed by 4-fold urinary hepcidin increases. RBT-3 up-regulated mouse hepcidin mRNA, but much more so in kidney (>25 times) versus liver (∼2 times). RBT-3 also activated kidney Nrf2 [increased Nrf2 nuclear binding; increased Nrf2-responsive gene mRNAs: heme oxygenase-1, sulfiredoxin-1, glutamate-cysteine ligase catalytic subunit and NAD(P)H quinone dehydrogenase 1]. RBT-3 preconditioning (18 h time lapse) markedly attenuated experimental cisplatin nephrotoxicity (∼50% blood urea nitrogen/creatinine decrements), in part by reducing renal cisplatin uptake by 40%. Exogenous hepcidin (without RBT-3) treatment conferred protection against mild in vivo (but not in vitro) cisplatin toxicity. RBT-3 acutely and dramatically up-regulates cytoprotective hepcidin production, increasing renal hepcidin levels. However, additional cytoprotective mechanisms are activated by RBT-3 (e.g. Nrf2 activation; reduced cisplatin uptake). Thus RBT-3-induced preconditioning likely confers renal resistance to cisplatin via an interplay of multiple cytoprotective activities.

Germacrone Reduces Cisplatin-Induced Toxicity of Renal Proximal Tubular Cells via Inhibition of Organic Cation Transporter.

Cisplatin is a widely used chemotherapy for solid tumors; however, its benefits are limited by serious nephrotoxicity, particularly in proximal tubular cells. The present study investigated the renoprotective effect and mechanisms of germacrone, a bioactive terpenoid compound found in Curcuma species on cisplatin-induced toxicity of renal cells. Germacrone (50 and 100 µM) attenuated apoptosis of human renal proximal tubular cells, RPTEC/TERT1 following treatment with 50 µM cisplatin and for 48 h. Co-treating RPTEC/TERT1 cells with cisplatin and germacrone significantly reduced cellular platinum content compared with cisplatin treatment alone. The effect of germacrone on organic cation transporter 2 (OCT2) which is a transporter responsible for cisplatin uptake was determined. Germacrone showed an inhibitory effect on OCT2-mediated methyl-4-phenylpyridinium acetate (3H-MPP+) uptake with IC50 of 15 µM with less effect on OCT1. The germacrone's protective effect on cisplatin-induced cytotoxicity was not observed in cancer cells; cisplatin's anti-cancer activity was preserved. In conclusion, germacrone prevents cisplatin-induced toxicity in renal proximal tubular cells via inhibition OCT2 transport function and reducing cisplatin accumulation. Thus germacrone may be a good candidate agent used for reducing cisplatin-induced nephrotoxicity.

A Multicompartment Human Kidney Proximal Tubule-on-a-Chip Replicates Cell Polarization-Dependent Cisplatin Toxicity.

Drug-induced kidney injury is a major clinical problem and causes drug attrition in the pharmaceutical industry. To better predict drug-induced kidney injury, kidney in vitro cultures with enhanced physiologic relevance are developed. To mimic the proximal tubule, the main site of adverse drug reactions in the kidney, human-derived renal proximal tubule epithelial cells (HRPTECs) were injected in one of the channels of dual-channel Nortis chips and perfused for 7 days. Tubes of HRPTECs demonstrated expression of tight junction protein 1 (zona occludens-1), lotus lectin, and primary cilia with localization at the apical membrane, indicating an intact proximal tubule brush border. Gene expression of cisplatin efflux transporters multidrug and toxin extrusion transporter (MATE) 1 (SLC47A1) and MATE2-k (SLC47A2) and megalin endocytosis receptor increased 19.9 ± 5.0-, 23.2 ± 8.4-, and 106 ± 33-fold, respectively, in chip cultures compared with 2-dimensional cultures. Moreover, organic cation transporter 2 (OCT2) (SLC22A2) was localized exclusively on the basolateral membrane. When infused from the basolateral compartment, cisplatin (25 µM, 72 hours) induced toxicity, which was evident as reduced cell number and reduced barrier integrity compared with vehicle-treated chip cultures. Coexposure with the OCT2 inhibitor cimetidine (1 mM) abolished cisplatin toxicity. In contrast, infusion of cisplatin from the apical compartment did not induce toxicity, which was in line with polarized localization of cisplatin uptake transport proteins, including OCT2. In conclusion, we developed a dual channel human kidney proximal tubule-on-a-chip with a polarized epithelium, restricting cisplatin sensitivity to the basolateral membrane and suggesting improved physiologic relevance over single-compartment models. Its implementation in drug discovery holds promise to improve future in vitro drug-induced kidney injury studies. SIGNIFICANCE STATEMENT: Human-derived kidney proximal tubule cells retained characteristics of epithelial polarization in vitro when cultured in the kidney-on-a-chip, and the dual-channel construction allowed for drug exposure using the physiologically relevant compartment. Therefore, cell polarization-dependent cisplatin toxicity could be replicated for the first time in a kidney proximal tubule-on-a-chip. The use of this physiologically relevant model in drug discovery has potential to aid identification of safe novel drugs and contribute to reducing attrition rates due to drug-induced kidney injury.