Intracellular Doxorubicin Concentration Research Articles

MiRNA-449 family is epigenetically repressed and sensitizes to doxorubicin through ACSL4 downregulation in triple-negative breast cancer

Despite progress in breast cancer treatment, a significant portion of patients still relapse because of drug resistance. The involvement of microRNAs in cancer progression and chemotherapy response is well established. Therefore, this study aimed to elucidate the dysregulation of the microRNA-449 family (specifically, microRNA-449a, microRNA-449b-5p, and microRNA-449c-5p) and its impact on resistance to doxorubicin, a commonly used chemotherapeutic drug for the treatment of triple-negative breast cancer. We found that the microRNA-449 family is downregulated in triple-negative breast cancer and demonstrated its potential as a diagnostic biomarker. Besides, our findings indicate that the downregulation of the microRNA-449 family is mediated by the microRNAs-449/SIRT1-HDAC1 negative feedback loop. Moreover, it was found that the microRNA-449 family dysregulates the fatty acid metabolism by targeting ACSL4, which is a potential prognostic biomarker that mediates doxorubicin response through regulation of the drug extrusion pump ABCG2. Altogether, our results suggest that the microRNA-449 family might be a potential therapeutic target for the treatment of triple-negative breast cancer since it is implicated in doxorubicin response through ACSL4/ABCG2 axis regulation. Ultimately, our results also highlight the value of microRNAs-449 and ACSL4 as diagnostic and prognostic biomarkers in triple-negative breast cancer.Proposed model of miRNAs-449 downregulation in TNBC and doxorubicin response. MiRNAs-449 are downregulated in TNBC through a negative feedback loop with SIRT1 and HDAC1. Moreover, ACSL4 increases ABCG2 expression, thus diminishing the intracellular doxorubicin concentration and promoting doxorubicin resistance. MiRNAs-449 overexpression downregulates the ACSL4/ABCG2 axis and sensitizes doxorubicin-resistant cells to doxorubicin. Created with BioRender. TNBC: triple-negative breast cancer; DOX: doxorubicin; SIRT1: Sirtuin 1; HDAC1: Histone deacetylase 1; ACSL4: Acyl-CoA Synthetase Long-Chain Family Member 4; ABCG2: ATP-binding cassette superfamily G member 2.

Modeling Doxorubicin Pharmacokinetics in Multiple Myeloma Suggests Mechanism of Drug Resistance.

Multiple myeloma (MM) is a plasma cell malignancy often treated with chemotherapy drugs. Among these, doxorubicin (DOXO) is commonly employed, sometimes in combined-drug therapies, but it has to be optimally administered in order to maximize its efficacy and reduce possible side effects. To support DOXO studies and treatment optimization, here we propose an experimental/modeling approach to establish a model describing DOXO pharmacokinetics (PK) in MM cells. A series of in vitro experiments were performed in MM1R and MOLP-2 cells. DOXO was administered at two dosages (200 nM, 450 nM) at [Formula: see text] = 0 and removed at [Formula: see text] = 3 hrs. Intracellular DOXO concentration was measured via fluorescence microscopy during both drug uptake ([Formula: see text] = 0-3 hrs) and release phases ([Formula: see text] = 3-8 hrs). Four PK candidate models were identified, and were compared and selected based on their ability to describe DOXO data and numerical parameter identification. The most parsimonious model consists of three compartments describing DOXO distribution between the extracellular space, the cell cytoplasm and the nucleus, and defines the intracellular DOXO efflux rate through a Hill function, simulating a threshold/saturation drug resistance mechanism. This model predicted DOXO data well in all the experiments and provided precise parameter estimates (mean ± standard deviation coefficient of variation: 15.8 ± 12.2%). A reliable PK model describing DOXO uptake and release in MM cells has been successfully developed. The proposed PK model, once integrated with DOXO pharmacodynamics, has the potential of allowing the study and the optimization of DOXO treatment strategies in MM.

A physiologically-based pharmacokinetic model for predicting doxorubicin disposition in multiple tissue levels and quantitative toxicity assessment

Doxorubicin is a widely-used chemotherapeutic drug, however its high toxicity poses a significant challenge for its clinical use. To address this issue, a physiologically-based pharmacokinetic (PBPK) model was implemented to quantitatively assess doxorubicin toxicity at cellular scale. Due to its unique pharmacokinetic behavior (e.g. high volume of distribution and affinity to extra-plasma tissue compartments), we proposed a modified PBPK model structure and developed the model with multispecies extrapolation to compensate for the limitation of obtaining clinical tissue data. Our model predicted the disposition of doxorubicin in multiple tissues including clinical tissue data with an overall absolute average fold error (AAFE) of 2.12. The model's performance was further validated with 8 clinical datasets in combined with intracellular doxorubicin concentration with an average AAFE of 1.98. To assess the potential cellular toxicity, toxicity levels and area under curve (AUC) were defined for different dosing regimens in toxic and non-toxic scenarios. The cellular concentrations of doxorubicin in multiple organ sites associated with commonly observed adverse effects (AEs) were simulated and calculated the AUC for quantitative assessments. Our findings supported the clinical dosing regimen of 75mg/m2 with a 21-day interval and suggest that slow infusion and separated single high doses may lower the risk of developing AEs from a cellular level, providing valuable insights for the risk assessment of doxorubicin chemotherapy. In conclusion, our work highlights the potential of PBPK modelling to provide quantitative assessments of cellular toxicity and supports the use of clinical dosing regimens to mitigate the risk of adverse effects.

A tumor-specific ROS self-supply enhanced cascade-responsive prodrug activation nanosystem for amplified chemotherapy against multidrug-resistant tumors.

Chemotherapy remains the mainstay of cancer treatment, and doxorubicin (DOX) is recommended as a first-line chemotherapy drug against cancer. However, systemic adverse drug reactions and multidrug resistance limit its clinical applications. Here, a tumor-specific reactive oxygen species (ROS) self-supply enhanced cascade responsive prodrug activation nanosystem (denoted as PPHI@B/L) was developed to optimize multidrug resistance tumor chemotherapy efficacy while minimizing the side effects. PPHI@B/L was constructed by encapsulating the ROS-generating agent β-lapachone (Lap) and the ROS-responsive doxorubicin prodrug (BDOX) in acidic pH-sensitive heterogeneous nanomicelles. PPHI@B/L exhibited particle size decrease and charge increase when it reached the tumor microenvironment due to acid-triggered PEG detachment, to favor its endocytosis efficiency and deep tumor penetration. Furthermore, after PPHI@B/L internalization, rapidly released Lap was catalyzed by the overexpressed quinone oxidoreductase-1 (NQO1) enzyme NAD(P)H in tumor cells to selectively raise intracellular ROS levels. Subsequently, ROS generation further promoted the specific cascade activation of the prodrug BDOX to exert the chemotherapy effects. Simultaneously, Lap-induced ATP depletion reduced drug efflux, synergizing with increased intracellular DOX concentrations to assist in overcoming multidrug resistance. This tumor microenvironment-triggered cascade responsive prodrug activation nanosystem potentiates antitumor effects with satisfactory biosafety, breaking the chemotherapy limitation of multidrug resistance and significantly improving therapy efficiency. STATEMENT OF SIGNIFICANCE: Chemotherapy remains the mainstay of cancer treatment, and doxorubicin (DOX) is recommended as a first-line chemotherapy drug against cancer. However, systemic adverse drug reactions and multidrug resistance limit its clinical applications. Here, a tumor-specific reactive oxygen species (ROS) self-supply enhanced cascade responsive prodrug activation nanosystem (denoted as PPHI@B/L) was developed to optimize multidrug resistance tumor chemotherapy efficacy while minimizing the side effects. The work provides a new sight for simultaneously addressing the molecular mechanisms and physio-pathological disorders to overcome MDR in cancer treatment.

Comparison of Characterization in Two-Dimensional and Three-Dimensional Canine Mammary Gland Tumor Cell Models.

Canine mammary gland tumors can be used as predictive models for human breast cancer. There are several types of microRNAs common in human breast cancer and canine mammary gland tumors. The functions of microRNAs in canine mammary gland tumors are not well understood. We compared the characterization of microRNA expression in two-dimensional and three-dimensional canine mammary gland tumor cell models. We evaluated the differences between two- and three-dimensional cultured canine mammary gland tumor SNP cells by assessing microRNA expression levels, morphology, drug sensitivity, and hypoxia. The expression of microRNA-210 in the three-dimensional-SNP cells was 10.19 times higher than that in the two-dimensional-SNP cells. The intracellular concentrations of doxorubicin in the two- and three-dimensional-SNP cells were 0.330 ± 0.013 and 0.290 ± 0.048 nM/mg protein, respectively. The IC50 values of doxorubicin for the two- and three-dimensional-SNP cells were 5.2 and 1.6 μM, respectively. Fluorescence of the hypoxia probe, LOX-1, was observed inside the sphere of three-dimensional-SNP cells without echinomycin but not in two-dimensional-SNP cells. The three-dimensional-SNP cells treated with echinomycin showed weak LOX-1 fluorescence. The present study showed a clear difference in microRNA expression levels in cells cultured in a two-dimensional adherent versus a three-dimensional spheroid model.

Discovery of a novel highly potent and low-toxic jatrophane derivative enhancing the P-glycoprotein-mediated doxorubicin sensitivity of MCF-7/ADR cells

Use of novel modulators targeting P-glycoprotein (P-gp, ABCB1 transporter) is among the most accepted strategies for overcoming multidrug resistance in cancer chemotherapy. In the current study, we pursued our structure-activity relationship studies of jatrophane derivatives by structural modification of compound 1, a natural jatrophane isolated from Euphorbia. sororia A. Nine compounds exhibited higher reversal activity in P-gp/ABCB1-mediated MCF-7/ADR cells than verapamil (VRP). The cytotoxicity and doxorubicin (DOX) intracellular accumulation effects of jatrophane derivatives were assessed in normal HEK293T cells and DOX-resistant MCF-7/ADR cells. The most potent compound 17 merits multiple activities, including (1) high efficiency (EC50=182.17±32.67nM) in reversing P-gp-mediated resistance to DOX, low cytotoxicity, and a high therapeutic index; and (2) increasing the accumulation of Rhodamine123 (Rho123) and DOX in a dose-dependent manner compared to verapamil in MCF-7/ADR cells. Our results indicated that the reversal activity of 17 was due to the stimulation of the P-gp ATPase activity instead of the direct inhibition of P-gp protein expression. A docking study demonstrated that 17 has a high binding affinity toward the DOX recognition site of P-gp. This resulted in 17 enhancing the sensitivity of DOX to MCF-7/ADR cells by stimulating P-gp ATPase activity, increasing intracellular DOX and Rho123 concentrations, inhibiting the phosphoinositide 3-kinase/serine-threonine kinase mediated by DOX and further reducing the expression of P-gp. This study provides a promising P-gp inhibitor for reversing multidrug resistance and provides a basis for further research.

Abstract 380: Tumor treating fields enhance cellular drug uptake in mesothelioma cell lines

Abstract Malignant pleural mesothelioma (MPM) is an aggressive malignancy with a 5-year survival rate of less than 10% and it remains a cancer lacking effective therapy options when tumors progress. The development of novel drug regimens for MPM treatment have been limited during the last two decades, with the exception of combinations including immune checkpoint inhibitors. In 2019, Tumor Treating Fields (TTFields) in combination with pemetrexed plus platinum-based chemotherapy were approved by the FDA for the first-line treatment of unresectable, locally advanced or metastatic, MPM. However, the mechanism of action of TTFields and their interaction with chemotherapeutic agents are not fully elucidated yet, thus a better knowledge may contribute to the design of more effective clinical trials for MPM patients. A panel of primary MPM cells was isolated from pleural effusion and/or lavages of patients’ thoracic cavity, before administration of therapeutic treatment, obtaining cell lines from the 3 subtypes of MPM. Epithelioid and sarcomatoid subtypes revealed different sensitivities to TTFields, with a stronger impact of the treatment on cell proliferation in the epithelioid subtype. This was associated with a higher modulation of transcription at different times of TTFields exposure, as demonstrated by RNA-seq analysis. Some of the genes modulated by the treatment encode membrane transport proteins, especially of the solute carrier family (SLC), in both sarcomatoid and epithelioid cells. This suggested a possible role of TTFields in cellular drug uptake and efflux. To test this hypothesis two cell lines, the sarcomatoid CD60 and the epithelioid CD473, have been treated with 150 kHz TTFields in combination with doxorubicin (DXR) following different schedules of treatment and the intracellular drug concentrations were assessed by HPLC measurements. TTFields exposure increased cellular uptake of DXR in both MPM models, but the effect was higher in CD473 epithelioid cells. After 24h of treatment with 1 µM DXR the amount of intracellular drug in sarcomatoid cells exposed to TTFields was almost twice that of the samples treated with DXR alone, while in epithelioid cells intracellular DXR concentration was almost four times higher than that found in TTFields untreated samples. This behavior might be ascribed to a different modulation of the gene coding for P-glycoprotein (ABCB1), which was markedly downregulated by TTFields treatment in epithelioid cells only. Similar experiments are in progress to test the impact of TTFields on cellular drug uptake in different combination schedules with other anticancer compounds. These data suggest that TTFields may increase the efficacy of drug treatments by enhancing the intracellular drug concentration and their possible confirmation by further experiments may represent an important rationale for the design of novel MPM therapies. Citation Format: Monica Lupi, Rosy Amodeo, Lavinia Morosi, Laura Mannarino, Lara Paracchini, Sergio Marchini, Federica Grosso, Roberta Libener, Giovanni L. Ceresoli, Maurizio D'Incalci. Tumor treating fields enhance cellular drug uptake in mesothelioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 380.

In Vitro Cell Toxicity and Intracellular Uptake of Doxorubicin Exposed as a Solution or Liposomes: Implications for Treatment of Hepatocellular Carcinoma.

Cytostatic effects of doxorubicin in clinically applied doses are often inadequate and limited by systemic toxicity. The main objective of this in vitro study was to determine the anti-tumoral effect (IC50) and intracellular accumulation of free and liposomal doxorubicin (DOX) in four human cancer cell lines (HepG2, Huh7, SNU449 and MCF7). The results of this study showed a correlation between longer DOX exposure time and lower IC50 values, which can be attributed to an increased cellular uptake and intracellular exposure of DOX, ultimately leading to cell death. We found that the total intracellular concentrations of DOX were a median value of 230 times higher than the exposure concentrations after exposure to free DOX. The intracellular uptake of DOX from solution was at least 10 times higher than from liposomal formulation. A physiologically based pharmacokinetic model was developed to translate these novel quantitative findings to a clinical context and to simulate clinically relevant drug concentration–time curves. This showed that a liver tumor resembling the liver cancer cell line SNU449, the most resistant cell line in this study, would not reach therapeutic exposure at a standard clinical parenteral dose of doxorubicin (50 mg/m2), which is serious limitation for this drug. This study emphasizes the importance of in-vitro to in-vivo translations in the assessment of clinical consequence of experimental findings.

The inhibition of ABCB1/MDR1 or ABCG2/BCRP enables doxorubicin to eliminate liver cancer stem cells

Two ATP-binding cassette transporters, ABCB1/MDR1 and ABCG2/BCRP, are considered the most critical determinants for chemoresistance in hepatocellular carcinoma. However, their roles in the chemoresistance in liver cancer stem cells remain elusive. Here we explored the role of inhibition of MDR1 or ABCG2 in sensitizing liver cancer stem cells to doxorubicin, the most frequently used chemotherapeutic agent in treating liver cancer. We show that the inhibition of MDR1 or ABCG2 in Huh7 and PLC/PRF/5 cells using either pharmacological inhibitors or RNAi resulted in the elevated level of intracellular concentration of doxorubicin and the accompanied increased apoptosis as determined by confocal microscopy, high-performance liquid chromatography, flow cytometry, and annexin V assay. Notably, the inhibition of MDR1 or ABCG2 led to the reversal of the chemoresistance, as evident from the enhanced death of the chemoresistant liver cancer stem cells in tumorsphere-forming assays. Thus, the elevation of effective intracellular concentration of doxorubicin via the inhibition of MDR1 or ABCG2 represents a promising future strategy that transforms doxorubicin from a traditional chemotherapy agent into a robust killer of liver cancer stem cells for patients undergoing transarterial chemoembolization.

Next generation risk assessment (NGRA): Bridging in vitro points-of-departure to human safety assessment using physiologically-based kinetic (PBK) modelling – A case study of doxorubicin with dose metrics considerations

Using the chemical doxorubicin (DOX), the objective of the present study was to evaluate the impact of dose metrics selection in the new approach method of integrating physiologically-based kinetic (PBK) modelling and relevant human cell-based assays to inform a priori the point of departure for human health risk. We reviewed the literature on the clinical consequences of DOX treatment to identify dosing scenarios with no or mild cardiotoxicity observed. Key concentrations of DOX that induced cardiomyocyte toxicity in vitro were derived from studies of our own and others. A human population-based PBK model of DOX was developed and verified against pharmacokinetic data. The model was then used to predict plasma and extracellular and intracellular heart concentrations of DOX under selected clinical settings and compared with in vitro outcomes, based on several dose metrics: Cmax (maximum concentration) or AUC (area under concentration-time curve) in free or total form of DOX. We found when using in vitro assays to predict cardiotoxicity for DOX, AUC is a better indicator. Our study illustrates that when appropriate dose metrics are used, it is possible to combine PBK modelling with in vitro-derived toxicity information to define margins of safety and predict low-risk human exposure levels.

Amino Acid Starvation Sensitizes Resistant Breast Cancer to Doxorubicin-Induced Cell Death.

Many clinical trials are beginning to assess the effectiveness of compounds known to regulate autophagy in patients receiving anti-cancer chemotherapy. However, autophagy inhibition, through exogenous inhibitors, or activation, through starvation, has revealed conflicting roles in cancer management and chemotherapeutic outcome. This study aimed to assess the effect of amino acid starvation on doxorubicin-treated breast cancer cells by assessing the roles of autophagy and apoptosis. An in vitro breast cancer model consisting of the normal breast epithelial MCF12A and the metastatic breast cancer MDAMB231 cells was used. Apoptotic and autophagic parameters were assessed following doxorubicin treatments, alone or in combination with bafilomycin, ATG5 siRNA or amino acid starvation. Inhibition of autophagy, through ATG5 siRNA or bafilomycin treatment, increased caspase activity and intracellular doxorubicin concentrations in MCF12A and MDAMB231 cells during doxorubicin treatment. While amino acid starvation increased autophagic activity and decreased caspase activity and intracellular doxorubicin concentrations in MCF12A cells, no changes in autophagic parameters or caspase activity were observed in MDAMB231 cells. Our in vivo data showed that 24 h protein starvation during high dose doxorubicin treatment resulted in increased survival of tumor-bearing GFP-LC3 mice. Results from this study suggest that short term starvation during doxorubicin chemotherapy may be a realistic avenue for adjuvant therapy, especially with regards to the protection of non-cancerous cells. More research is however, needed to fully understand the regulation of autophagic flux during starvation.

Despite Blocking Doxorubicin-Induced Vascular Damage, Quercetin Ameliorates Its Antibreast Cancer Activity.

Quercetin is a naturally occurring flavonol present in many foods. Doxorubicin is an effective anticancer agent despite its dose-limiting cardiovascular toxicity. Herein, we investigated the potential protective effects of quercetin against doxorubicin-induced vascular toxicity and its effect on the therapeutic cytotoxic profile of doxorubicin in breast cancer cell lines. The incubation of isolated aortic rings with doxorubicin produced concentration-dependent exaggeration of vasoconstriction responses to phenylephrine but impaired vasodilation responses to acetylcholine. Coincubation with quercetin completely blocked the exaggerated vasoconstriction responses and the impaired vasodilation. In addition, doxorubicin incubation increased reactive oxygen species generation from the isolated aorta, while coincubation with quercetin inhibited ROS generation back to normal values. On the other hand, quercetin in combination with doxorubicin, doubled the IC50 of doxorubicin alone in MCF-7 cells from 0.4 ± 0.03 to 0.8 ± 0.06 μM. To a lesser extent, the IC50 of doxorubicin did not change after combination with quercetin in MDA-MB-231 cells. These findings indicate a significant antagonistic interaction between quercetin and doxorubicin in the aforementioned cell lines. Only in T47D cells, quercetin combination with doxorubicin was an additive interaction (CI − value = 1.17). Yet, quercetin significantly impaired the immediate phase of intracellular ROS generation by doxorubicin within breast cancer cells from 125.2 ± 3.6% to 102.5 ± 3.9% of control cells. Using annexin-V/FITC staining technique, the quercetin/doxorubicin combination showed a significantly lower percent of apoptotic cells compared to doxorubicin alone treated cells. Cell cycle distribution in breast cancer cells was performed using DNA content flowcytometry after propidium iodide staining. Quercetin induced significant accumulation of cells in the S phase as well as in the G2/M phase within both MCF-7 and MDA-MB-231 cell lines and interfered with doxorubicin-induced cell cycle effects. Interestingly, quercetin was found to inhibit the P-glycoprotein ATPase subunit with a consequent enhanced intracellular concentration of doxorubicin in MDA-MB-231 and T47D cells. In conclusion, quercetin, despite its potent vascular protective activity against doxorubicin, was found to influence doxorubicin-induced antibreast cancer effects via pharmacodynamic as well as cellular pharmacokinetic aspects.

The \u03b2-adrenergic receptor antagonist propranolol offsets resistance mechanisms to chemotherapeutics in diverse sarcoma subtypes: a pilot study

Standard chemotherapy for soft tissue sarcomas has shown limited efficacy. Here, we sought to evaluate whether β-adrenergic receptor (β-AR) signalling contributed to the progression of sarcomas and therapy resistance. To assess the translational potential of β-adrenergic receptors, we performed immunohistochemical detection of β1-AR, β2-AR and β3-AR in leiomyosarcoma, liposarcoma and angiosarcoma tissue specimens, reporting the results scored for the intensity. By using established and patient-derived sarcoma cells, we demonstrated the antitumour potential of the pharmacological targeting of β-ARs with the nonselective β-blocker propranolol in such sarcomas. Of note, pharmacological β-AR inhibition synergized with doxorubicin in inhibiting the cell viability of liposarcoma and leiomyosarcoma cells and increased the response to docetaxel in angiosarcoma- and solitary fibrous tumour (SFT)-patient-derived cells. Notably, the SFT patient was treated with the combination of propranolol and docetaxel, reporting prolonged disease control. Mechanistically, we found that propranolol reduced the activity of the multidrug resistance efflux pump P-gp, thereby increasing the intracellular doxorubicin concentration and antitumour activity. In addition, propranolol attenuated the Akt-dependent survival signal induced by doxorubicin and strongly reduced the activation of the NF-kB/COX-2 pathway, increasing cell sensitivity to docetaxel. Overall, our study highlighted the therapeutic potential of propranolol, alone or in rational combination therapies, for sarcoma treatment.

Charge-reversal ZnO-based nanospheres for stimuli-responsive release of multiple agents towards synergistic cancer therapy

Abstract 2,3-Dimethylmaleic anhydride (DMMA)-decorated zinc oxide (ZnO) nanoparticles with doxorubicin (DOX) and phenylsulfonyl furoxan (PSF) loaded were designed and prepared through a step-by-step strategy. Amino-terminated ZnO nanoparticles were synthesized, in which DOX was encapsulated by forming coordination bonding between DOX and Zn2+ ions. PSF as a NO donor was conjugated to the nanoparticles via amide bonding. Surface decoration of DMMA endowed the resultant (DOX,PSF)@ZnO-DMMA nanospheres with charge-reversal ability, as characterized by zeta potential measurements. The (DOX,PSF)@ZnO-DMMA nanospheres (an average size of ~7.9 nm) had a mildly negative surface charge and thus had a long blood circulation. The intratumoral microenvironment could reverse the surface charge of the nanospheres, which favored the cellular uptake of the nanospheres. Decomposition of ZnO occurred under acidic conditions characteristic of intracellular endosomal and lysosomal systems, leading to release of both Zn2+ ions and DOX. In the meantime, NO release from the PSF was triggered by the high concentration of GSH in the cancerous cells. The nanospheres were able to inhibit the proliferation of cancerous cells efficiently, as evidenced by in vitro cell assay and in vivo small animal experiments. The NO greatly reduced the multi-drug resistance (MDR) and increased the intracellular concentration of DOX, which killed cancerous cells in combination with the Zn2+ ions. This work has highlighted the delivery of multiple therapeutic agents in response to intracellular stimuli, which offered a new approach for rational design of nanomaterials towards synergistic cancer therapy.

Tumor‐Oriented Telomerase‐Terminated Nanoplatform as Versatile Strategy for Multidrug Resistance Reversal in Cancer Treatment

Multidrug resistance is one of the major problems in chemotherapy, and exploiting impactful targets to reverse drug resistance of most tumors remains a difficult problem. In this study, the tumor-oriented nanoparticle, BIBR1532-loaded peptide dendrimeric prodrug nanoassembly (B-PDPN), is used to assist telomerase inhibition for multidrug resistance reversal. B-PDPN possesses the characteristics of an acid-activated histidine to promote cellular uptake, a redox-sensitive poly(ethylene glycol) (PEG) layer to actualize endosomal escape and telomerase inhibitor release, and an acid sensitive chemical bond to facilitate chemotherapeutic drug release. Telomerase termination weakens the protective effect of hTERT protein on mitochondria and enhances reactive oxygen species (ROS) production, which increases DNA damage and apoptosis. The tumor-oriented nanoparticle B-PDPN achieves a broad-spectrum telomerase inhibition to combat multidrug resistance. In vivo experiments support the evidence that B-PDPN accumulates in the tumor site and reduces the expression of hTERT in tumor tissues to inhibit drug resistant tumor growth. This work introduces an innovative strategy of utilizing features of tumor-activated nanoplatform to assist telomerase termination. The nanoplatform enhances intracellular drug concentration and nucleus delivery of doxorubicin (DOX), and promotes DNA damage to combat multidrug resistance.

Canagliflozin inhibits p-gp function and early autophagy and improves the sensitivity to the antitumor effect of doxorubicin

Cancer easily induces resistance to most chemotherapy drugs. In this study, we investigated the combination cytotoxic and antitumor effects of canagliflozin (CAN) and doxorubicin (DOX) in vitro and in vivo. CAN significantly increased the cytotoxicity of DOX in HepG2, HepG2-ADR (adriamycin or doxorubicin-resistant) and MCF7 cells. CAN significantly promoted the intracellular uptake of DOX in HepG2 cells. CAN also reduced the P-glycoprotein (P-gp) level in HepG2 cells. The function of P-gp required ATP, but CAN significantly reduced the intracellular ATP level. CAN might inhibit the function of p-gp, increase the intracellular DOX concentration and contribute to an enhanced cytotoxic activity. Autophagy plays a protective role in chemotherapy-induced cell survival. However, CAN significantly inhibited DOX-induced autophagy in HepG2 cells, and the mechanism appeared to be mediated by promoting ULK1 ser 757 phosphorylation. The downregulation of P-gp may be associated with protein degradation but is independent of the autophagy pathway. Furthermore, in HepG2-xenograft BALB/c nude mice, CAN significantly increased the antitumor effect of DOX. This study is the first to report that a classical antidiabetic drug, CAN improved the sensitivity to the antitumor effect of DOX, and the potential molecular mechanisms of CAN may involve the inhibition of P-gp function and the autophagy pathway.

β1-Integrin binding to collagen type 1 transmits breast cancer cells into chemoresistance by activating ABC efflux transporters

Molecular interactions of tumor cells with the microenvironment are regarded as onset of chemotherapy resistance, referred to as cell adhesion mediated drug resistance (CAM-DR). Here we elucidate a mechanism of CAM-DR in breast cancer cells in vitro. We show that human MCF-7 and MDA-MB-231 breast cancer cells decrease their sensitivity towards cisplatin, doxorubicin, and mitoxantrone cytotoxicity upon binding to collagen type 1 (COL1) or fibronectin (FN). The intracellular concentrations of doxorubicin and mitoxantrone were decreased upon cell cultivation on COL1, while cellular cisplatin levels remained unaffected. Since doxorubicin and mitoxantrone are transporter substrates, this refers to ATP binding cassette (ABC) efflux transporter activities. The activation of the transporters BCRP, P-gp and MRP1 was shown by fluorescence assays to distinguish the individual input of these transporters to resistance in presence of COL1 and related to their expression levels by western blot. An ABC transporter inhibitor was able to re-sensitize COL1-treated cells for doxorubicin and mitoxantrone toxicity. Antibody-blocking of β1-integrin (ITGB1) induced sensitization towards the indicated cytostatic drugs by attenuating the increased ABC efflux activity. This refers to a key role of ITGB1 for matrix binding and subsequent transporter activation. A downregulation of α2β1 integrin following COL1 binding appears as clear indication for the relationship between ITGB1 and ABC transporters in regulating resistance formation, while knockdown of ITGB1 leads to a significant upregulation of all three transporters. Our data provide evidence for a role of CAM-DR in breast cancer via an ITGB1 - transporter axis and offer promising therapeutic targets for cancer sensitization.

Abstract 3633: Evauation of doxorubicin-thanine co-loaded nanoparticles for cancer chemotherapy

Abstract [Purpose] Novel nanoparticles have the potential to increase the efficacy of cancer chemotherapy. Recently, the use of two chemotherapeutic drugs load to liposomes has been studied. In particular, cytarabine-daunorubicin (1:5) co-loaded liposome (CPX-351) has high therapeutic efficacy against acute myelocytic leukemia. We previously reported that a combination of theanine (a specific amino acid of green tea) with doxorubicin (DOX) had a stronger antitumor effect than DOX alone. The effect is caused by the competitive inhibition of glutamate transport by theanine as a glutamate derivative via glutamate transporters in tumor cells. Subsequently, decreased glutamate levels in tumor cells result in lower glutathione, which makes efflux of DOX from cells difficult. Thus, it was speculated that specific accumulation of DOX, caused by theanine, increases the therapeutic index. In the present study, we examined the availability of a DOX-theanine co-loaded liposome as drug carrier for cancer chemotherapy. [Methods] DOX-theanine co-loaded liposome (DT-lip) was prepared using the thin-lipid method. DOX and theanine were loaded at the lipid membrane layer and internal water layer, respectively. P388 leukemia cells were used to study the influence of DT-lip on DOX influx and efflux. The cell suspension was incubated with DT-lip at 37°C, and the DOX concentration in tumor cells was determined. In the antitumor effect study in vivo, tumor-bearing mice were intravenously administered DT-lip (DOX 2.5 mg/kg) at 17th, 20th, and 23rd days after tumor cell implant. Tumors and normal tissue were removed 48 h after last administration. Tumor weight and DOX concentration in tissue were measured. [Results and Discussion] DT-lip was prepared with three ratios of DOX to theanine (1:0.5, 1:1, and 1:4, w/w). In the influx study, the DOX concentration in tumor cells was the same for the different ratios. DOX influx into tumor cells in the DT-lip group was enhanced compared to liposomes loaded with DOX only (D-lip). At 30 min, the intracellular DOX concentration in the DT-lip was 1.4 fold higher than that in the D-lip group. Conversely, there was no practical difference in DOX efflux from tumor cells between the D-lip and DT-lip group. In other words, DT-lip had an influence on influx system, and this phenomenon differed from the previous results for theanine′s role in the DOX efflux system. This findings suggest that liposomalization of theanine + DOX changed the theanine connected integrated mechanism in tumor cells. In vivo, the antitumor effect of DT-lip was superior to combined administration of both solutions. The adverse effects of DT-lip treatment on normal tissue did not increase because DOX concentration in tissue was comparable across group. In conclusion, the DT-lip, a liposome co-loaded DOX and theanine, is a novel antitumor formulation for cancer chemotherapy. Citation Format: Ikumi Sugiyama, Yasuyuki Sadzuka. Evauation of doxorubicin-thanine co-loaded nanoparticles for cancer chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3633.

Chemotherapy of Breast Cancer Cells Using Novel pH-Responsive Cellulose-Based Nanocomposites.

Purpose: The objective of the current study was to compare the anticancer efficacy of doxorubicin-loaded cellulose based magnetic (Fe3O4), zinc oxide (ZnO) nanoparticles on and free doxorubicin (DOX) on MCF-7 breast cancer cells. Methods: Novel pH-sensitive cellulose-graft poly acrylic acid based Fe3O4 (Cellulose-g-PAAg- PAcMNPs) and ZnO (Cellulose-g-PAA-g-PAcZnO) nanocomposites were synthesized via polymerization of acrylic acid and modified 3-(trimethoxysilyl) propyl methacrylate onto the cellulosic backbone via reversible addition-fragmentation chain transfer (RAFT) method. Results: Cellulose-g-PAA-g-PAcMNPs and Cellulose-g-PAA-g-PAcZnO nanocarriers with mean diameter of 15 and 38 nm were prepared successfully. DOX was loaded effectively to the ZnO and Fe3O4 nanocarriers via complexing and electrostatic force with great encapsulation efficiency of 99.07% and 98.92%, respectively. DOX-loaded nanocarriers showed obvious pHdependent tumor specific drug release pattern. MTT assay results indicated that IC50 of the DOX loaded Cellulose-g-PAA-g-PAcZnO, DOX loaded Cellulose-g-PAA-g-PAcMNPs and free DOX after 48 hours treatment with MCF7 cell lines were about 24.03, 49.27 and 99.76 μg mL−1, respectively. Therefore both DOX nanoformulations significantly increase antitumor ability compared to free DOX (P < 0.05). The results of MTT assay and DAPI staining revealed that DOX-loaded Cellulose-g-PAA-g-PAcZnO NPs show higher chemotherapy efficiency in MCF7 breast cancer cell line compare to the DOX-loaded Cellulose-g-PAA-g-PAcMNPs due to high interaction of ZnO with DOX. Conclusion: The formation of the complexes between the DOX and ZnO nanoparticles at the chelating sites of the quinone and the phenolic oxygen molecules of DOX, lead to more sustained drug release and enhanced chemotherapy effectiveness by increasing the intracellular concentration of DOX.

LevistolideA synergistically enhances doxorubicin‑induced apoptosis of k562/dox cells by decreasing MDR1 expression through the ubiquitin pathway.

Multidrug resistance (MDR) is one of the main reasons underlying failure of cancer chemotherapy. Certain natural compounds may help prevent MDR, and may be used in combination with chemotherapeutic agents to enhance their efficacy. Levistolide A is a natural product that is extracted from the rhizome of Angelicae sinensis (Oliv.), which has been used as an essential component of antitumor formulas since ancient times in China. The present study conducted the following experiments: MTT assay, apoptosis analysis, cellular doxorubicin accumulation assay, immunoblotting and reverse transcription‑quantitative polymerase chain reaction, to investigate whether levistolide A enhance doxorubicin‑induced apoptosis of k562/dox cells and to determine the molecular mechanisms involved. When combined with doxorubicin, levistolide A exhibited a synergistic effect and induced cytotoxicity in k562/dox cells. Drug accumulation studies revealed that levistolide A increased the intracellular concentration of doxorubicin in a dose‑dependent manner. Cell apoptosis experiments indicated that levistolide A increased the sensitivity of k562/dox cells to doxorubicin. Furthermore, detection of reactive oxygen species (ROS) revealed that levistolide A enhanced doxorubicin‑induced cell death by increasing the levels of ROS. Mitochondrial potential detection with JC‑1 staining also indicated that levistolide A synergistically enhanced doxorubicin‑induced cell death. Immunoblotting demonstrated that levistolide A enhanced doxorubicin‑induced cell death by decreasing the expression levels of B‑cell lymphoma 2 and increasing caspase 3 expression. Furthermore, multidrug resistance protein 1 (MDR1) expression in k562/dox cells was downregulated by levistolide A in a dose‑dependent manner, thus suggesting that levistolide A may modulate MDR1 during cancer therapy. Therefore, the combination of levistolide A with doxorubicin could result in more effective and less toxic anticancer regimens.