Expression Level Of P-glycoprotein Research Articles

Mitotic Arrest Deficient 2 Like 1 Contributes to Colorectal Cancer Cell Migration, Invasion, and Oxaliplatin Resistance Through the Wnt/β-Catenin Pathway.

Colorectal cancer (CRC) is a highly prevalent malignancy, requiring chemotherapy for advanced stages of the disease. Previously, we found that mitotic arrest deficient 2 like 1 (MAD2L1) was upregulated and facilitated malignant proliferation in CRC. However, the association between MAD2L1 expression and tumor progression, as well as chemotherapy resistance in CRC, remains unclear. The progression capacities of CRC cells were assessed using transwell and wound healing assays, and the resistance to cisplatin in oxaliplatin-resistant CRC cells was assessed using CCK-8 assay and flow cytometry. Relevant protein levels of epithelial-to-mesenchymal transition (EMT) and Wnt/β-catenin pathway were analyzed using western blotting. Revealing the impact of MAD2L1 on metastasis and drug resistance in CRC through inhibition of the Wnt/β-catenin pathway. Knockdown of MAD2L1 attenuated the malignant progression of CRC cells, inhibited EMT, and blocked the Wnt/β-catenin pathway. MAD2L1 was significantly upregulated in oxaliplatin-resistant CRC cells, accompanied by the activation of the Wnt/β-catenin pathway. Knockdown of MAD2L1 effectively reversed oxaliplatin resistance, leading to apoptosis and downregulation of the protein expression levels of β-catenin, P-glycoprotein (P-gp), and ABCG2. After the knockdown of MAD2L1, the inhibition of the Wnt/β-catenin pathway exhibited a synergistic effect, effectively suppressing malignant progression and reversing oxaliplatin resistance in CRC cells. So, knockdown of MAD2L1 suppressed cell malignant progression, equally sensitized resistant CRC cells to oxaliplatin, potentially by blocking the activation of the Wnt/β-catenin pathway.

Induction of cytochrome P450 via upregulation of CAR and PXR: a potential mechanism for altered florfenicol metabolism by macranthoidin B in vivo.

Macranthoidin B (MB) is a primary active component of Flos Lonicerae. In Chinese veterinary clinics, Flos Lonicerae is frequently used in combination with florfenicol to prevent and treat infections in livestock and poultry. However, potential interactions between Flos Lonicerae and florfenicol remain unclear. To systematically study these interactions, it is crucial to investigate the individual phytochemicals within Flos Lonicerae. Therefore, MB was selected for this study to assess its effect on the pharmacokinetics of florfenicol in vivo and to explore the underlying mechanisms involved. Male Sprague-Dawley rats were administered MB (60 mg/kg BW) or sterile water orally for 7 consecutive days. On the 8th day, a single oral dose of florfenicol (25 mg/kg BW) was given. Florfenicol pharmacokinetics were analyzed using ultra-high performance liquid chromatography. The hepatic expression levels of cytochrome P450 (CYP1A2, CYP2C11, CYP3A1), UDP-glucuronosyltransferase (UGT1A1), P-glycoprotein (P-gp), and nuclear receptors, including constitutive androstane receptor (CAR), pregnane X receptor (PXR), and retinoid X receptor alpha (RXRα), were quantified via reverse transcription-quantitative polymerase chain reaction and Western blotting (WB). Hepatic CYP1A2 and CYP2C11 activities were measured using a cocktail method. Additionally, the subcellular expression and localization of CAR, PXR, and RXRαin hepatocytes was assessed using WB and immunofluorescence staining. MB significantly reduces the AUC(0-∞) and MRT(0-∞) of florfenicol. MB also markedly upregulates the mRNA and protein expression of hepatic CYP1A2 and CYP2C11, along with their catalytic activities. Substantial upregulation of CAR and PXR proteins occurs in the hepatocyte nucleus, along with significant nuclear colocalization of the transcriptionally active CAR/RXRα and PXR/RXRαheterodimers, indicating MB-induced nuclear translocation of both CAR and PXR. These findings suggest that MB-induced alterations in florfenicol pharmacokinetics, particularly its accelerated elimination, may be due to increased expression and activities of CYP1A2 and CYP2C11, with CAR and PXR potentially involved in these regulatory effects. Further investigation is yet needed to fully elucidate the clinical implications of these interactions concerning the efficacy of florfenicol in veterinary medicine.

Tibetan tea extract exerts anti-tumor effects and potentiate paclitaxel-induced cytotoxicity and apoptosis in human hepatocellular carcinoma cells HepG2

Tibetan Tea (TT), traditionally esteemed by indigenous communities on the Qinghai-Tibet Plateau for over thirteen centuries, is a post-fermented tea recognized for its wide spectrum of biological functions. These include antioxidant capabilities, hypocholesterolemic effects, anti-hyperglycemic properties, and immune system enhancement. Nevertheless, additional medicinal potentials of TT, including its anticancer effects, remain to be unveiled in empirical studies. We aimed to evaluate the antiproliferative and chemotherapy-enhancing effects of TT extract on human hepatocellular carcinoma (HCC) cells, thereby initiating an inquiry into the potential molecular mechanisms involved. In this study, it was observed that TT extract suppressed the proliferation of HCC cells and triggered significant apoptosis. This apoptotic induction was facilitated through the activation of caspase-3, caspase-8, and caspase-9, along with decreased expression ratio of Bcl-2/Bax protein in HCC cells. Moreover, the combinational treatment of TT extract and Paclitaxel (Taxol) was found to enhance the anticancer effect. The expression level of P-glycoprotein (P-gp) encoded by the MDR1 (Multiple Drug Resistance) gene was remarkably decreased, revealing that TT extract might have the potential to be utilized as an adjuvant to promote HCC treatment. In conclusion, the Tibetan Tea extract exhibits cytotoxic, anticancer, and chemosensitization properties.

Chemosensitizing effect of pentoxifylline in sensitive and multidrug-resistant non-small cell lung cancer cells.

Aim: Multidrug resistance (MDR) is frequent in non-small cell lung cancer (NSCLC) patients, which can be due to its fibrotic stroma. This work explores the combination of pentoxifylline, an anti-fibrotic and chitinase 3-like-1 (CHI3L1) inhibitor drug, with conventional chemotherapy to improve NSCLC treatment. Methods: The effect of pentoxifylline in the expression levels of P-glycoprotein (P-gp), CHI3L1 and its main downstream proteins, as well as on cell death, cell cycle profile, and P-gp activity was studied in two pairs of sensitive and MDR counterpart NSCLC cell lines (NCI-H460/NCI-H460/R and A549/A549-CDR2). Association studies between CHI3L1 gene expression and NSCLC patients' survival were performed using The Cancer Genome Atlas (TCGA) analysis. The sensitizing effect of pentoxifylline to different drug regimens was evaluated in both sensitive and MDR NSCLC cell lines. The cytotoxicity of the drug combinations was assessed in MCF10A non-tumorigenic cells. Results: Pentoxifylline slightly decreased the expression levels of CHI3L1, β-catenin and signal transducer and activator of transcription 3 (STAT3), and caused a significant increase in the G1 phase of the cell cycle in both pairs of NSCLC cell lines. A significant increase in the % of cell death was observed in the sensitive NCI-H460 cell line. TCGA analysis revealed that high levels of CHI3L1 are associated with low overall survival (OS) in NSCLC patients treated with vinorelbine. Moreover, pentoxifylline sensitized both pairs of sensitive and MDR NSCLC cell lines to the different drug regimens, without causing significant toxicity to non-tumorigenic cells. Conclusion: This study suggests the possibility of combining pentoxifylline with chemotherapy to increase NSCLC therapeutic response, even in cases of MDR.

Cationic anticancer peptide L-K6-modified and doxorubicin-loaded mesoporous silica nanoparticles reverse multidrug resistance of breast cancer

Multidrug resistance (MDR) is the major challenge for chemotherapy of cancer and makes the treatment benefits unsustainable. To overcome this issue, nanotechnology has been introduced and various nanocarriers have been constructed to reverse MDR in drug resistant cancer cells. Here, mesoporous silica nanoparticle (MSN-COOH) were modified with cationic anticancer peptide L-K6 (MSN@L-K6), and further loaded with doxorubicin (DOX) to construct an anticancer nano-system, MSN@L-K6@DOX. Our results showed that MSN@L-K6@DOX released DOX in pH-sensitive manner, which enable to be highly efficient in delivering drugs to tumors, counterbalancing the MDR. In addition, MSN@L-K6@DOX treatment decreased the expression level of P-glycoprotein in MCF-7/ADR cells, enhanced the intracellular accumulation of DOX, and thereafter reversed the MDR of MCF-7/ADR cells to DOX. Moreover, the MDR reversal activity of MSN@L-K6@DOX was further confirmed in xenograft mouse model, as evidenced by the decreased tumor volume and weight. These results indicate the MDR reversal activity of MSN@L-K6@DOX and may provide a novel strategy for the development of nano-based drug delivery system to overcome cancer MDR.

MiR-34 by targeting p53 induces apoptosis and DNA damage in paclitaxel-resistant human oral squamous carcinoma cells.

MicroRNA-34 (miR-34) is one the most important tumor suppressor miRNAs involving in the various aspects of oral cancer. The present study aimed to evaluate the effects of miR-34 restoration in OECM-1 oral cancer resistant to paclitaxel (OECM-1/PTX) and its underlying mechanisms through p53-mediated DNA damage and apoptosis. OECM-1 and OECM-1/PTX were transfected with miR-34 mimic and inhibitor. Cellular proliferation and apoptosis were evaluated through MTT assay and flow cytometry, respectively. The mRNA and protein expression levels of p53, p-glycoprotein (P-gp), ATM, ATR, CHK1, and CHK2 were assessed through qRT-PCR and western blotting. Rhodamin123 uptake assay was used to measure the P-gp activities. P53 expression was also suppressed by sing a siRNA transfection of cells. The expression levels of miR-34 were downregulated in OECM-1/PTX. Restoration of miR-34 led to increase in cytotoxic effects of paclitaxel in cells. In addition, the expression levels and activities of P-gp were reduced following miR-34 transfection. miR-34 transfection upregulated the p53, ATM, ATR, CHK1, and CHK2 expression levels in OECM-1/PTX cells. Furthermore, cells transfected with miR-34 showed higher levels of apoptosis. miR-34 restoration reverses paclitaxel resistance in OECM-1 oral cancer. The chemosensitive effects of miR-34 is mediated through increasing DNA damage and apoptosis in a p53 depended manner.

Calcium Phosphate-Based Nanoformulation Selectively Abolishes Phenytoin Resistance in Epileptic Neurons for Ceasing Seizures.

Phenytoin (PHT) is a first-line antiepileptic drug in clinics, which could decrease neuronal bioelectric activity by blocking the voltage-operated sodium channels. However, the intrinsically low blood-brain-barrier (BBB)-crossing capability of PHT and upregulated expression level of the efflux transporter p-glycoprotein (P-gp) coded by the gene Abcb1 in epileptic neurons limit its efficacy in vivo. Herein, a nanointegrated strategy to overcome PHT resistance mechanisms for enhanced antiepileptic efficacy is reported. Specifically, PHT is first incorporated into calcium phosphate (CaP) nanoparticles through biomineralization, followed by the surface modification of the PEGylated BBB-penetrating TAT peptide. The CaP@PHT-PEG-TAT nanoformulation could effectively cross the BBB to be taken in by epileptic neurons. Afterward, the acidic lysosomal environment would trigger their complete degradation to release Ca2+ and PHT into the cytosol. Ca2+ ions would inhibit mitochondrial oxidative phosphorylation to reverse cellular hypoxia to block hypoxia-inducible factor-1α (Hif1α)-Abcb1-axis, as well as disrupt adenosine triphosphate generation, leading to simultaneous suppression of the expression and drug efflux capacity of P-gp to enhance PHT retention. This study offers an approach for effective therapeutic intervention against drug-resistant epilepsy.

Wine-processed Chuanxiong Rhizoma enhances efficacy of aumolertinib against EGFRmutant non-small cell lung cancer xenografts in nude mouse brain

To investigate the effect of wine-processed Chuanxiong Rhizoma (WCR) for enhancing the efficacy of aumolertinib against xenografts of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) in the brain of nude mice. In a co-culture system of hCMEC/D3 and PC9 NSCLC cells, the effect of aqueous extract of WCR (2 mg/mL) combined with aumolertinib (10 and 20 μmol/L) on apoptosis of PC9 cells was investigated using flow cytometry. The effects of WCR extract (0.5, 1, and 2 mg/mL) on transmembrane transport of 8 μmol/L aumolertinib was examined in ABCB1-MDCK monolayer cells. Western blotting was used to detect the expressions of the tight junction proteins related with blood- brain barrier integrity. A nude mouse model bearing NSCLC xenograft in the brain was established to observe the inhibitory effect of WCR (1 mg/g) combined with aumolertinib (10 mg/kg) on tumor growth. Compared with aumolertinib (20 μmol/L) alone, WCR extract (2 mg/mL) combined with aumolertinib significantly increased the apoptosis rate of PC9 cells by 21% (P < 0.01). The combined treatment with WCR (0.5, 1, 2 mg/mL) obviously increased apical-basolateral transport of aumolertinib in ABCB1-MDCK monolayer cells (P < 0.05) and significantly lowered the expression levels of zonula occludens-1, claudin-5 and P-glycoprotein (P < 0.05). In the tumor-bearing mice, compared with aumolertinib alone, the combined treatment with WCR and aumolertinib produced stronger inhibitory effect on tumor growth, improved weight loss, and prolonged the survival time of the nude mice (P < 0.05). Pathological examination showed that the combined treatment obviously increased the apoptosis rate of the tumor cells and alleviated neural injuries in the brain. Immunohistochemistry revealed that WCR treatment significantly reduced the expressions of ZO-1 and claudin-5 in the brain of the mice. WCR combined with aumolertinib shows stronger inhibitory effects against tumor xenografts of EGFR-mutant NSCLC possibly due to the effect of WCR in facilitating the transmembrane transport of aumolertinib by downregulating ZO-1, claudin-5 and P-glycoprotein expression.

Expression of GnT-III decreases chemoresistance via negatively regulating P-glycoprotein expression: Involvement of the TNFR2-NF-κB signaling pathway

The phenomenon of multidrug resistance (MDR) is called chemoresistance with respect to the treatment of cancer, and it continues to be a major challenge. The role of N-glycosylation in chemoresistance, however, remains poorly understood. Here, we established a traditional model for adriamycin resistance in K562cells, which are also known as K562/adriamycin-resistant (ADR) cells. Lectin blot, mass spectrometry, and RT-PCR analysis showed that the expression levels of N-acetylglucosaminyltransferase III (GnT-III) mRNA and its products, bisected N-glycans, are significantly decreased in K562/ADR cells, compared with the levels in parent K562cells. By contrast, the expression levels of both P-glycoprotein (P-gp) and its intracellular key regulator, NF-κB signaling, are significantly increased in K562/ADR cells. These upregulations were sufficiently suppressed by the overexpression of GnT-III in K562/ADR cells. We found that the expression of GnT-III consistently decreased chemoresistance for doxorubicin and dasatinib, as well as activation of the NF-κB pathway by tumor necrosis factor (TNF) α, which binds to two structurally distinct glycoproteins, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), on the cell surface. Interestingly, our immunoprecipitation analysis revealed that only TNFR2, but not TNFR1, contains bisected N-glycans. The lack of GnT-III strongly induced TNFR2's autotrimerization without ligand stimulation, which was rescued by the overexpression of GnT-III in K562/ADR cells. Furthermore, the deficiency of TNFR2 suppressed P-gp expression while it increased GnT-III expression. Taken together, these results clearly show that GnT-III negatively regulates chemoresistance via the suppression of P-gp expression, which is regulated by the TNFR2-NF/κB signaling pathway.

PH-/Redox-Responsive Nanodroplet Combined with Ultrasound-Targeted Microbubble Destruction for the Targeted Treatment of Drug-Resistant Triple Negative Breast Cancer.

Multiple drug resistance (MDR) exists in divergent cancers including triple negative breast cancer (TNBC) and partly results in the resistance to many first-line anti-cancer agents, bringing a big challenge to TNBC management. To develop novel TNBC therapeutics, in our study, a hyaluronic acid (HA)-carboxymethyl chitosan (CMC) conjugate linked via a disulfide-bond (HA-SS-CMC, HSC) was synthesized to fabricate nanodroplets (NDs). The NDs encapsulating doxorubicin (DOX) and perfluorohexane (DOX-HSC-NDs) were prepared via a homogenization/emulsification strategy and exhibited not only high biocompatibility but also noticeable tumor cell targeting ability and dual pH/redox responsiveness. Besides, DOX-HSC-NDs can be used as a contrast-enhanced ultrasound imaging agent for specific tumor imaging. DOX-HSC-NDs in combination with ultrasound targeted microbubble destruction could improve intracellular drug aggregation and retention of MDR cells and work against multiple mechanisms of drug resistance through synergistic strategies, including up-regulating the reactive oxygen species (ROS) level, promoting apoptosis, and scavenging glutathione, while reducing the expression levels of P-glycoprotein and inhibiting the epithelial-mesenchymal transition. This combination strategy showed protective effects against TNBC in both MDA-MB-231/ADR cells and tumor-bearing mice. Our study for the first time developed and reported the ultrasound-augmented HSC-NDs as the DOX nanocarrier and provided scientific evidence to support the future application of DOX-HSC-NDs as a potential TNBC therapy.

Discovery of benzochromene derivatives first example with dual cytotoxic activity against the resistant cancer cell MCF-7/ADR and inhibitory effect of the P-glycoprotein expression levels

A series of 1H-benzo[f]chromene moieties (4a–z) were synthesised under Ultrasonic irradiation and confirmed with spectral analyses. Derivative 4i solely possessed an X-ray single crystal. The anti-proliferative efficacy of the desired molecules has been explored against three cancer cells: MCF-7, HCT-116, and HepG-2 with the cytotoxically active derivatives screened against MCF-7/ADR and normal cells HFL-1 and WI-38. Furthermore, compounds 4b–d, 4k, 4n, 4q, and 4w, which possessed good potency against MCF-7/ADR, were tested as permeability glycoprotein (P-glycoprotein [P-gp]) expression inhibitors. The attained data confirmed that 4b–d, 4q, and 4w exhibited strong expression inhibition against the P-gp alongside its cytotoxic effect on MCF-7/ADR. The western blot results and Rho123 accumulation assays showed that compounds 4b–d, 4q, and 4w effectively inhibited the P-gp expression and efflux function. Meanwhile, 4b–d, 4q, and 4w induced apoptosis and accumulation of the treated MCF-7/ADR cells in the G1 phase and 4k and 4n in the S phase of the cell cycle.

Effect of Cyclooxygenase Inhibition on P-Glycoprotein Expression and Phenytoin Level in Brain Tissue of Pilocarpine Induced Epilepsy in Rats

Effect of Cyclooxygenase Inhibition on P-Glycoprotein Expression and Phenytoin Level in Brain Tissue of Pilocarpine Induced Epilepsy in Rats

Natural borneol serves as an adjuvant agent to promote the cellular uptake of piperlongumine for improving its antiglioma efficacy

Piperlongumine (PL) can selectively inhibit the proliferation of various cancer cells by increasing reactive oxygen species (ROS) level to cause a redox imbalance in cancer cells rather than in normal cells. However, the clinical application of PL is limited by its poor cellular uptake. Natural borneol (NB) is extracted from the fresh branches and leaves of Cinnamomum camphora (L.) Presl. with the purity of (+)-borneol no less than 96.0%. NB has been often used as an adjuvant agent to promote the cellular uptake of other drugs. This study aims to investigate the effect of NB on the cellular uptake of PL for improving its antiglioma efficacy and underlying mechanism. NB obviously promoted the cellular uptake of PL with a 1.3-fold increase in the maximum peak concentration and an earlier peak time of 30 min in C6 glioma cells. The cellular uptake of PL was enhanced by NB through down-regulating the expression levels of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2). The combination of NB and PL significantly induced higher levels of ROS, which increased apoptosis and enhanced G2/M cycle arrest of C6 glioma cells, compared to PL alone administration. NB-enhanced antiglioma efficacy of PL without side effects was confirmed in tumor-bearing mice, which was attributed to the improved cellular uptake of PL. The distribution of PL in the tumor tissue of combined group increased 2.39 times than that of PL-treated group. We firstly report NB as an adjuvant agent to improve the antiglioma efficacy of PL in a ROS-dependent manner, which is due to the enhanced cellular uptake of PL by NB though down-regulating the expression levels of ABCB1 and ABCG2. This work provides a new strategy to promote the cellular uptake of PL with great potential for the treatment of glioma.

Ethoxysanguinarine directly targets CIP2A to inhibit proliferation and induce autophagy of SGC7901/DDP cells

This study aims to investigate the effect of ethoxysanguinarine(Eth) on cisplatin(DDP)-resistant human gastric cancer cells and decipher the underlying mechanism. The human gastric cancer cell line SGC7901 and the DDP-resistant cell line SGC7901/DDP were used as the cell models. Western blot was employed to determine the expression levels of multidrug resistance-related proteins, and methyl thiazolyl tetrazolium(MTT) assay to detect the proliferation of SGC7901 and SGC7901/DDP cells exposed to DDP. After treatment with different concentrations of Eth, the proliferation of SGC7901 and SGC7901/DDP cells was detected by MTT assay, trypan blue exclusion assay, colony formation assay, and high-content imaging and analysis system. The apoptosis of SGC7901/DDP cells was detected by flow cytometry with Annexin V-FITC/PI staining. GFP-LC3 transfection was carried out to detect the effect of Eth on the autophagy of SGC7901/DDP cells. The expression levels of the multidrug resistance-related protein P-glycoprotein(P-gp), the apoptosis-related proteins [caspase-9, caspase-3, and poly(ADP-ribose) polymerase(PARP)], the autophagy-related protein light chain 3-Ⅱ(LC3-Ⅱ), the key effectors [mammalian target of rapamycin(mTOR), 70 kDa ribosomal protein S6 kinase(P70 S6 K), and 4 E binding protein 1(4 E-BP1)] of the mammalian target of rapamycin complex 1(mTORC1) signaling pathway, cancerous inhibitor of protein phosphatase 2A(CIP2A), and protein kinase B(Akt) were measured by Western blot. The mRNA level of CIP2A in the SGC7901/DDP cells exposed to Eth for 24 h was analyzed by RT-qPCR. After SGC7901/DDP cells were transfected with CIP2A expression vector pcDNA3.1-HA-CIP2A and treated with different concentrations of Eth, MTT assay was used to determine the prolife-ration of SGC7901/DDP cells and Western blot to detect the expression levels of related proteins. The interaction sites of Eth and CIP2A were predicted by molecular docking. The affinity between Eth and CIP2A was determined by drug affinity responsive target stability(DARTS) assay. The pharmacokinetic properties and drug-like activity of Eth were predicted by SwissADME. The results indicated that SGC7901/DDP cells were more sensitive to Eth than SGC7901 cells. Eth significantly inhibited proliferation and colony formation and changed the morphology, roundness, and area of SGC7901/DDP cells. Eth treatment caused the nucleus shrinking and significantly increased the apoptosis rate of the cells. Furthermore, Eth down-regulated the expression of caspase-9 and caspase-3 precursors and promoted the cleavage of PARP, which suggested that Eth induced the apoptosis of SGC7901/DDP cells. The GFP-LC3 in Eth-treated cells showed speckled aggregation. The up-regulated expression of LC3-Ⅱ by Eth indicated that Eth activated the autophagy of SGC7901/DDP cells. Eth down-regulated the expression of P-gp, the phosphorylation of mTOR, P70 S6K, and 4E-BP1, the expression of CIP2A, and the phosphorylation of Akt. Additionally, it increased the activity of PP2A, and had no significant effect on the expression of CIP2A in SGC7901/DDP cells. CIP2A overexpression antagonized the inhibition of cell proliferation and the activation of autophagy by Eth. Molecular docking suggested that Eth bound to CIP2A. The results of DARTS assay further proved the above binding effect. Eth has potential drug-like activity. The above results demonstrated that Eth inhibited the proliferation, induced the apoptosis, and activated the autophagy of SGC7901/DDP cells by targeting CIP2A and then down-regulating PP2A/mTORC1 signaling pathway. This study provided a new target for the treatment of cisplatin-resistant gastric cancer.

Hsa_circ_0000098 is a novel therapeutic target that promotes hepatocellular carcinoma development and resistance to doxorubicin

BackgroundCircular RNA (circRNA) is crucial to the progression of hepatocellular cancer (HCC). In addition, Mitochondrial calcium uniporter regulatory factor 1 (MCUR1) is commonly overexpressed in HCC to increase cellular ATP levels. Due to the highly aggressive characteristics of HCC, it is essential to identify new diagnostic biomarkers and therapeutic targets that may facilitate the diagnosis of HCC and the development of effective anti-HCC treatments.MethodsA series of in vitro and in vivo experiments were undertaken to investigate the biological importance and underlying mechanisms of circ_0000098 in HCC.ResultsThe expression of circ_0000098 was higher in HCC tissues compared to paired adjacent tissues. According to the receiver-operating characteristic curves, circ_0000098 functioned as a potential diagnostic tumor marker in HCC. Our experiments indicated that circ_0000098 served as a key oncogenic circRNA to increase HCC cell proliferation and invasion in vitro and HCC progression in vivo. Furthermore, mechanistic investigation demonstrated that by sequestering miR-383 from the 3′-UTR of MCUR1, circ_0000098 positively regulated MCUR1 expression in HCC cells and finally promoted HCC progression. On the other hand, inhibiting circ_0000098 in HCC cells could diminish doxorubicin (DOX) resistance by decreasing P-glycoprotein (P-gp, MDR1) expression and intracellular ATP levels. Either downregulation of MCUR1 or overexpression of miR-383 improved DOX sensitivity in HCC cells. Subsequently, a short hairpin RNA targeting circ_0000098 (referred to as sh-1) and doxorubicin (DOX) were encapsulated into platelets (PLTs), referred to as DOX/sh-1@PLT. Activated DOX/sh-1@PLT through HCC cells resulted in the creation of platelet-derived particles that were capable of delivering the DOX/sh-1 combination into HCC cells and promoting intracellular DOX accumulation. Furthermore, our in vivo experiments showed that DOX/sh-1@PLT can effectively reduce P-gp expression, promote DOX accumulation, and reverse DOX resistance.ConclusionsOur results demonstrated that circ_0000098 is an oncogenic circRNA that promotes HCC development through the miR-383/MCUR1 axis and targeting circ_0000098 with DOX/sh-1@PLT may be a promising and practical therapeutic strategy for preventing DOX resistance in HCC.

In Vitro Safety, Off-Target and Bioavailability Profile of the Antiviral Compound Silvestrol.

We characterized the in vitro safety and bioavailability profile of silvestrol, a compound effective against various viruses, such as corona- and Ebolaviruses, with an EC50 value of about 5 nM. The cytotoxic profile of silvestrol was assessed in various cancer cell lines, as well as the mutagenic and genotoxic potential with Ames and micronuclei tests, respectively. To identify off-target effects, we investigated whether silvestrol modulates G-protein coupled receptor (GPCR) signaling pathways. To predict the bioavailability of silvestrol, its stability, permeability and cellular uptake were determined. Silvestrol reduced viability in a cell-type-dependent manner, mediated no off-target effects via GPCRs, had no mutagenic potential and minor genotoxic effects at 50 nM. Silvestrol did not disturb cell barrier integrity, showed low membrane permeability, was stable in liver microsomes and exhibited good cellular uptake. Efficient cellular uptake and increased cytotoxicity were observed in cell lines with a low expression level of the transport protein P-glycoprotein, the known efflux transporter of silvestrol. In conclusion, silvestrol showed low permeability but good cellular uptake and high stability. Cell-type-dependent cytotoxicity seems to be caused by the accumulation of silvestrol in cells lacking the ability to expel silvestrol due to low P-glycoprotein levels.

Characterization of the Newly Established Homoharringtonine- (HHT-) Resistant Cell Lines and Mechanisms of Resistance.

Homoharringtonine- (HHT-) based HHT, aclarubicin, and cytarabine (HAA) induction regimen is the first-line therapy for nonelder acute myeloid leukemia (AML) patients in China. However, drug resistance is a new challenge, and little attention has been devoted to excavating resistant mechanisms. This study used the classic method to construct six HHT-resistant cell lines with a gradually increasing resistance index (RI) to discover HHT drug resistance mechanisms dynamically. After HHT resistance, the cell growth rate decreased, cell cycle delayed, and P-glycoprotein (p-gp, CD243) expression levels increased. Furthermore, we explored the changes in transcriptomics between HHT-sensitive and HHT-resistant cells using RNA-sequence. Through Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and hub gene analyses, we found that immune activity, especially G-protein coupled receptor (GPR) and related molecules, may mediate HHT resistance. Moreover, Calcitonin Receptor-Like (CALCRL) and G Protein Subunit Alpha I1 (GNAI1), which belong to GPRs, were stimulated in HHT-resistant cell strains in vitro and vivo, indicating that they may play a critical role in HHT resistance. In addition, these two genes have prognostic significance for AML patients. Taken together, we successfully constructed HHT-resistant cell lines with dynamic RIs and explored the resistance mechanisms, which will help identify new drugs for HHT-resistant AML patients.

Effect of Vildagliptin on the Level of P-Glycoprotein Expression in Normal Conditions and Alloxan–Induced Type 2 Diabetes Mellitus

INTRODUCTION: Vildagliptin is one of the drugs of choice in the treatment of type 2 diabetes mellitus (DM). In the literature review, no study has reported the effect of vildagliptin on the expression level of the transporter protein P-glycoprotein (P-gp, ABCB1 protein).&#x0D; AIM: To assess the in vivo effects of vildagliptin on the level of Р-gp expression in normal conditions and experimental alloxan-induced type 2 DM.&#x0D; MATERIALS AND METHODS: Type 2 DM was induced by a single intravenous injection of freshly prepared alloxan monohydrate solution in citrate buffer (pH = 4.0) at a dose of 80 mg/kg. Glucose content (mmol/L) was determined by the glucose oxidase method using Human kit (Germany) and insulin content (U/mL) by radioimmunoassay technique using Immunotech kit (Czech Republic). As an express method for the determination of glucose levels in the whole blood, OneTouch UltraEasy electrochemical glucose monitor (USA) was used. The level of P-gp expression in rabbit tissues (i.e., jejunum, liver, kidney, and cerebral cortex) was evaluated by the indirect histochemical method. After preliminary treatment, the histological material was embedded in paraffin. Antigens were retrieved before the immune-staining reaction. Then, the sections were incubated with primary antibodies to P-gp (ABCB1 antibody, middle region; 0.1 mL [Aviva Systems Biology ARP51326-P050, USА]). For immune staining, a polymer detection system with a peroxidase label (Leica Microsystems, Germany) was used. The cell nuclei were stained with hematoxylin. Photos of the micropreparation were captured using Canon Power Shot G5 digital camera with 400 magnification. Digital images were analyzed and processed using ImageJ and IHC Profiler plugin designed for quantitative immunohistochemical analysis. The results obtained from healthy animals, animals with alloxan-induced DM, healthy animals administered vildagliptin, and animals with modeled DM administered vildagliptin were compared.&#x0D; RESULTS: After day 14 of administration to healthy rabbits and day 5 of vildagliptin discontinuation (5 mg/kg), no statistically significant changes in P-gp expression in the aforementioned tissues and the levels of basal and postprandial insulin and blood glucose were noted. After the 14-day therapy of alloxan-induced type 2 DM with vildagliptin (5 mg/kg), a statistically significant recovery of expression of the P-gp expression in the jejunum, liver, and kidney was reduced due to the underlying pathology. In addition, the values of healthy rabbits and statistically significant normalization of the insulin and blood glucose levels were observed. On day 5 of drug cancellation, significant inhibition of P-gp expression in the jejunum, liver, and kidney was noted again, and there was a significant reduction of postprandial insulin and blood glucose. No significant alterations of ABCB1 protein expression in the hematoencephalic barrier were noted during the studied periods.&#x0D; CONCLUSION: The administration of vildagliptin (5 mg/kg) to rabbits with alloxan-induced type 2 DM promotes the recovery of P-gp expression in the jejunum, liver, and kidney, which were reduced due to the underlying pathology. Meanwhile, its administration to healthy animals had no significant effect on the expression of P-gp.

Knockout of P-glycoprotein abolish the collateral sensitivity of CHORC5 multidrug resistant cells

Multidrug resistant tumor cells show collaterally sensitive to a range of non-toxic drugs. In this report, we describe the isolation of several P-glycoprotein-knockout cell clones, using CRISPR/Cas9, from Chinese hamster multidrug resistant model cell line and its parental cells (e.g., CHORC5 and AuxB1, respectively). All three P-glycoprotein-knockout clones of CHORC5 cells show complete loss of resistance to anti-cancer drugs (e.g., colchicine and doxorubicin), while gaining resistance to well characterized collateral sensitivity drugs (e.g., verapamil, progesterone and NSC73306). A correlation between P-glycoprotein and Sorcin expression levels and a possible role for the latter in low grade resistance to colchicine and doxorubicin was observed. Furthermore, we show that P-glycoprotein expression is necessary for the ROS-mediated mechanism of collateral sensitivity. However, expectantly, P-glycoprotein-knockout clones of CHORC5 cells revealed a dramatic increase in the accumulation of Rhodamine 123, Mito tracker red and doxorubicin, but not Hoechst 33342. The latter findings and their significance to P-glycoprotein collateral sensitivity remain to be determined.

Biomarkers to target and silence stemness of breast cancer stem cell model: silencing MDR1 by siRNA

Abstract Objectives Aim of the study was to reveal new biomarker genes to target breast cancer stem-like cells (BCSC-like) and then sensitize BCSC-like cells to chemotherapy by silencing MDR1 gene found to be the most suitable target. Methods Drug resistance associated genes were screened by cDNA microarray to unveil biomarker genes in drug resistance breast cancer model cells. Drug resistance was then reversed by silencing MDR1 gene in BCSC-like cells. The effect of silencing was monitored by real-time cell proliferation analysis. Differential expressions of MDR1, ALDH1A3, EGFR and BAG4 genes were identified by real-time PCR. P-glycoprotein (P-gp) expression level and its activity were investigated by Western blot and flow cytometry measurements, respectively. Results 16 new biomarker genes were identified upon gene expression analysis by cDNA microarray. MDR1 gene was selected as the most potent target gene and silencing of it caused down-regulation of MDR1, ALDH1A3, EGFR, BAG4 expression and P-glycoprotein activity and expression in BCSC-like cells. At the end, silenced BCSC-like cells were found to be more responsive to paclitaxel therapy. Conclusions In conclusion, siMDR1 silencing is an effective way to reverse multidrug resistance and malignancy. New biomarker genes revealed in this study require to be investigated to target stemness of BC.