Accumulation Of Rhodamine Research Articles

P-gp modulating effect of Azadirachta indica extract in multidrug-resistant cancer cell lines.

The extract of Azadirachta indica, commonly known as neem, has found extensive use in traditional medicine for treating various human diseases. In this study, the effect of the 50% ethanol extract of A. indica (AI01) on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) was examined using MDR cell lines, specifically paclitaxel-resistant HepG2 (PR-HepG2) and doxorubicin (DOX)-resistant (DR) colon-26 cells. 96-h treatment of the two cell lines with AI01 (30 μg/mL) showed no effect on the expression of P-gp mRNA (human MDR1 and mouse mdr1b) and protein, while AI01 increased the accumulation of rhodamine 123, a P-gp substrate, in both PR-HepG2 and DR-colon-26 cells. The cytotoxic effects of 48-h treatment with AI01 on the viability of PR-HepG2 and DR-colon-26 cells were not observed. Therefore, 30 μg/mL AI01 may have no cytotoxic and P-gp-inducing effects. Finally, AI01 potentiated the sensitivity of PR-HepG2 and DR-colon-26 cell lines to DOX by 8.6- and 15.3-fold, respectively. These findings suggest that A. indica may be a promising source for a new class of P-gp modulators without cytotoxic/P-gp induction effects.

Ceritinib Enhances the Efficacy of Substrate Chemotherapeutic Agent in Human ABCB1-Overexpressing Leukemia Cells In Vitro, In Vivo and Ex-Vivo

Background/Aims: Multidrug resistance (MDR) triggered by ATP binding cassette (ABC) transporters, such as ABCB1, ABCC1, and ABCG2, is a key obstacle for successful cancer chemotherapy. There is currently no FDA-approved MDR modulator that can be used in clinic. Ceritinib, a selective ALK inhibitor, has been approved as the second-line treatment for ALK-positive non-small cell lung cancer. Here, we examined the role of ceritinib in leukemia associated MDR in therapy. Methods: The cell proliferation was detected by MTT assay. The flow cytometry was used to detect the expression of cell surface protein and to detect the accumulation and efflux of rhodamine 123 (Rh123) or doxorubicin (Dox) in cells. The RT-PCR and Western blot were performed to detect the gene expression and protein expression levels, respectively. Results: We found that ceritinib enhanced the efficacy of substrate chemotherapeutic agent in ABCB1-overexpressing K562/adr leukemia cells both in vitro and in vivo models, but neither in sensitive parental K562 leukemia cells nor in ABCC1-overexpressing HL-60/adr leukemia cells. Mechanistically, ceritinib significantly increased the intracellular accumulation of Rh123 or Dox but did neither alter ABCB1 expressions at both protein and mRNA levels nor block the phosphorylations of AKT and ERK1/2 at the concentration of MDR reversal. Importantly, ceritinib also increased the intracellular accumulation of Dox and enhanced the efficacy of Dox in primary leukemia cells in ex-vivo. Conclusion: Our results suggested that ceritinib enhanced the efficacy of substrate chemotherapeutic agent on inhibition of leukemia cell growth in vitro, in vivo and ex-vivo, which linked to block ABCB1 function, pumping out its substrate conventional chemotherapeutic agent, thereby increasing the intracellular accumulation. These suggest the combination of ceritinib and substrate chemotherapeutic drugs maybe an effective treatment of resistant leukemia patients with ABCB1-mediated MDR.

Reversal of multidrug resistance by icaritin in doxorubicin-resistant human osteosarcoma cells

Multidrug resistance (MDR) is one of the major obstacles in cancer chemotherapy. Our previous study has shown that icariin could reverse MDR in MG-63 doxorubicin-resistant (MG-63/DOX) cells. It is reported that icariin is usually metabolized to icariside II and icaritin. Herein, we investigated the effects of icariin, icariside II, and icaritin (ICT) on reversing MDR in MG-63/DOX cells. Among these compounds, ICT exhibited strongest effect and showed no obvious cytotoxicity effect on both MG-63 and MG-63/DOX cells ranging from 1 to 10 μmol·L−1. Furthermore, ICT increased accumulation of rhodamine 123 and 6-carboxyfluorescein diacetate and enhanced DOX-induced apoptosis in MG-63/DOX cells in a dose-dependent manner. Further studies demonstrated that ICT decreased the mRNA and protein levels of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). We also verified that blockade of STAT3 phosphorylation was involved in the reversal effect of multidrug resistance in MG-63/DOX cells. Taken together, these results indicated that ICT may be a potential candidate in chemotherapy for osteosarcoma.

SH003 reverses drug resistance by blocking signal transducer and activator of transcription 3 (STAT3) signaling in breast cancer cells.

Overcoming drug resistance is an important task for investigators and clinician to achieve successful chemotherapy in cancer patients. Drug resistance is caused by various factors, including the overexpression of P-glycoprotein (P-gp, MDR1). The development of new, useful compounds that overcome drug resistance is urgent. SH003 is extracted from the mixture of three different herbs, and its anticancer effect has been revealed in different cancer cell types. In the present study, we investigated whether SH003 is able to reverse drug resistance using paclitaxel-resistant breast cancer cells (MCF-7/PAC). In our experiments, SH003 significantly decreased cell growth and colony formation in MCF-7/PAC cells and parental MCF-7 cells. This growth inhibition was related to the accumulation of cells in the sub-G0/G1 apoptotic population and an increase in the number of apoptotic cells. SH003 reduced the mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated proteins (MRPs) in MCF-7/PAC cells. SH003 also down-regulated the expression of P-gp. SH003 reversed drug efflux from MCF-7/PAC cells, resulting in rhodamine123 (Rho123) accumulation. Inhibition of drug resistance by SH003 is related to the suppression of the signal transducer and activator of transcription 3 (STAT3) signaling pathway. SH003 decreased STAT3 activation (p-STAT3) and its nuclear translocation and inhibited the secretion of VEGF and MMP-2, which are STAT3 target genes. An STAT3 inhibitor, JAK inhibitor I and an HIF-1α inhibitor decreased cell growth in MCF-7 and MCF-7/PAC cells. Taken together, these results demonstrate that SH003 can overcome drug resistance, and SH003 might be helpful for chemotherapy in cancer patients.

Ethyl lucidenates A reverses P-glycoprotein mediated vincristine resistance in K562/A02 cells

Multidrug resistance is a major unresolved obstacle to successful cancer chemotherapy. It is often associated with an elevated efflux of a variety of anticancer drugs by ATP-binding cassette transporters including P-glycoprotein, BCRP and MRP1. In this study, the reversal effect of Ethyl lucidenates A on K562/A02 cells was investigated. At concentrations of 10 μM, Ethyl lucidenates A could reverse the resistance of K562/A02 to vincristine up to 7.59 folds. Mechanistically, Ethyl lucidenates A could increase the intracellular accumulation of vincristine in K562/A02 cells through inhibiting the P-glycoprotein mediated drug-transport activity by rhodamine accumulation assay and cell cycle analysis. Further mechanistic investigation found that Ethyl lucidenates A did not alter P-glycoprotein expression. In conclusion, Ethyl lucidenates A could reverse the multidrug resistance of K562/A02 cells via its influence on P-glycoprotein drug-transport activity and thus, be a potential multidrug resistance reversal agent.

In silico comparisons between natural inhibitors of ABCB1/P-glycoprotein to overcome doxorubicin-resistance in the NCI/ADR-RES cell line

To investigate compound-protein binding mode and molecular dynamic simulation of P-glycoprotein (P-gp), in silico studies were performed to compare 12 naturally occurring compounds using two softwares. The net results showed that piperine (PIP) had the best binding affinity. In vitro studies on doxorubicin (DOX)-resistant NCI/ADR-RES cells, known to express P-gp, showed that, dose-dependently, PIP significantly increased intracellular accumulation of rhodamine-123 and had cytotoxic effects accessed by MTT assay. In addition, PIP at 25 and 50μM significantly potentiated DOX-induced cytotoxicity on the same cell line. P-gp ATPase assay showed that both DOX and PIP had dose-dependent inhibition of orthovandate-sensitive ATPase activity, indicating they are both P-gp inhibitors, with IC50 of 84±1 and 37±2μM, respectively. PIP did not show any activation of ATPase activity, while DOX did, indicating that P-gp does not accept PIP as a substrate. Using DOX at concentration 33.33μM together with PIP (100μM), DOX-mediated P-gp ATPase activity was decreased to levels 4-folds lower than DOX alone. In conclusion, both in silico and in vitro studies confirm that PIP is an inhibitor of P-gp mediated DOX efflux, suggesting PIP as a promising adjuvant to DOX cancer chemotherapy.

In vitro screening of dual flavonoid combinations for reversing P-glycoprotein-mediated multidrug resistance: Focus on antiepileptic drugs

The combined use of different P-glycoprotein (P-gp) inhibitors may be a relevant approach to the synergistic and safer inhibition of the P-gp-mediated drug efflux. Herein, we aimed to explore dual combinations of the flavonoids baicalein, (-)-epigallocatechin gallate, kaempferol, quercetin and silymarin to reverse the interference of P-gp on the intracellular accumulation of antiepileptic drugs (AEDs). The intracellular accumulation of rhodamine 123 (a classic P-gp substrate) and of several commonly used AEDs (carbamazepine, phenytoin, oxcarbazepine) or their metabolites (carbamazepine-10,11-epoxide and licarbazepine) was evaluated in MDCK-MDR1 cells in the presence and absence of individual flavonoids and their combinations. A selected flavonoid combination [(-)-epigallocatechin gallate/silymarin] was also evaluated in transepithelial transport experiments using licarbazepine (active metabolite of oxcarbazepine) as a model compound. Most flavonoid combinations increased rhodamine 123 intracellular uptake in a greater extent than their additive individual effects at similar concentrations. Moreover, selected (-)-epigallocatechin gallate/silymarin and kaempferol/baicalein combinations also enhanced the intracellular accumulation of all AEDs and metabolites. Overall, the combination of (-)-epigallocatechin gallate/silymarin was the most promising one. Thus, dual flavonoid combinations may be useful to overcome the P-gp-mediated efflux of AEDs and their metabolites, making their association to AED therapy a potentially valuable approach to circumvent pharmacoresistance in epilepsy.

Tomentodione M sensitizes multidrug resistant cancer cells by decreasing P-glycoprotein via inhibition of p38 MAPK signaling.

In this study, we investigated the mechanism by which tomentodione M (TTM), a novel natural syncarpic acid-conjugated monoterpene, reversed multi-drug resistance (MDR) in cancer cells. TTM increased the cytotoxicity of chemotherapeutic drugs such as docetaxel and doxorubicin in MCF-7/MDR and K562/MDR cells in a dose- and time-dependent manner. TTM reduced colony formation and enhanced apoptosis in docetaxel-treated MCF-7/MDR and K562/MDR cells, and it enhanced intracellular accumulation of doxorubicin and rhodamine 123 in MDR cancer cells by reducing drug efflux mediated by P-gp. TTM decreased expression of both P-gp mRNA and protein by inhibiting p38 MAPK signaling. Similarly, the p38 MAPK inhibitor SB203580 reversed MDR in cancer cells by decreasing P-gp expression. Conversely, p38 MAPK-overexpressing MCF-7 and K562 cells showed higher P-gp expression than controls. These observations indicate that TTM reverses MDR in cancer cells by decreasing P-gp expression via p38 MAPK inhibition.

In vitroselection ofPhytomonas serpenscells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

Flavonoid compounds as reversing agents of the P-glycoprotein-mediated multidrug resistance: An in vitro evaluation with focus on antiepileptic drugs

The pharmacoresistance to antiepileptic drugs (AEDs) remains a major unsolved therapeutic need. The overexpression of multidrug transporters, as the P-glycoprotein (P-gp), at the level of the blood-brain barrier of epileptic patients has been suggested as a key mechanism underlying the refractory epilepsy. Thus, efforts have been made to search for therapeutically useful P-gp inhibitors. Herein, the strategy of flavonoid/AED combined therapy was exploited as a possible approach to overcome the P-gp-mediated pharmacoresistance. For this purpose, several in vitro studies were performed using Madin-Darby canine kidney II (MDCK II) cells and those transfected with the human multidrug resistance-1 (MDR1) gene, overexpressing the P-gp (MDCK-MDR1). Overall, the results showed that baicalein, (−)-epigallocatechin gallate, kaempferol, quercetin and silymarin, at 200μM, produced a marked increase on the intracellular accumulation of rhodamine 123 in MDCK-MDR1 cells, potentially through inhibiting the P-gp activity. In addition, with the exception of lamotrigine, all other AEDs tested (phenytoin, carbamazepine and oxcarbazepine) and their active metabolites (carbamazepine-10,11-epoxide and licarbazepine) demonstrated to be P-gp substrates. Furthermore, the most promising flavonoids as P-gp inhibitors promoted a significant increase on the intracellular accumulation of the AEDs (excluding lamotrigine) and their active metabolites in MDCK-MDR1 cells, evidencing to be important drug candidates to reverse the AED-resistance. Thus, the co-administration of AEDs with baicalein, (−)-epigallocatechin gallate, kaempferol, quercetin and silymarin should continue to be explored as adjuvant therapy for refractory epilepsy.List of chemical compounds studied in this article:Baicalein (PubChem CID: 5,281,605); Carbamazepine (PubChem CID: 2554); Carbamazepine 10,11-epoxide (PubChem CID: 2555); (−)-Epigallocatechin gallate (PubChem CID: 65064); Kaempferol (PubChem CID: 5280863); Lamotrigine (PubChem CID: 3878); Licarbazepine (PubChem CID: 114709); Oxcarbazepine (PubChem CID: 34312); Phenytoin (PubChem CID: 1775); Silymarin (PubChem CID: 7073228); Quercetin (PubChem CID: 5280343); Verapamil (PubChem CID: 2520).

Cholecalciferol-PEG ConjugateBased Nanomicelles of Doxorubicin for Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the leading cancer in women. Chemotherapeutic agents used for TNBC are mainly associated with dose-dependent toxicities and development of resistance. Hence, novel strategies to overcome resistance and to offer dose reduction are warranted. In this study, we designed a novel dual-functioning agent, conjugate of cholecalciferol with PEG2000 (PEGCCF) which can self-assemble into micelles to encapsulate doxorubicin (DOX) and act as a chemosensitizer to improve the therapeutic potential of DOX. DOX-loaded PEGCCF (PEGCCF-DOX) micelles have particle size, polydispersity index (PDI), and zeta potential of 40±8.7nm, 0.180±0.051, and 2.39±0.157mV, respectively. Cellular accumulation studies confirmed that PEGCCF was able to concentration-dependently enhance the cellular accumulation of DOX and rhodamine 123 in MDA-MB-231 cells through its P-glycoprotein (P-gp) inhibition activity. PEGCCF-DOX exhibited 1.8-, 1.5-, and 2.9-fold enhancement in cytotoxicity of DOX in MDA-MB-231, MDA-MB-468, and MDA-MB-231DR (DOX-resistant) cell lines, respectively. Western blot analyses showed that PEGCCF-DOX caused significant reduction in tumor markers including mTOR, c-Myc, and antiapoptotic marker Bcl-xl along with upregulation of preapoptotic marker Bax. Further, reduction in mTOR activity by PEGCCF-DOX indicates reduced P-gp activity due to P-gp downregulation as well and, hence, PEGCCF causes enhanced chemosensitization and induces apoptosis. Substantially enhanced apoptotic activity of DOX (10-fold) in MDA-MB-231(DR) cells confirmed apoptotic potential of PEGCCF. Conclusively, PEGCCF nanomicelles are promising delivery systems for improving anticancer activity of DOX in TNBC, thereby reducing its side effects and may act as a potential carrier for other chemotherapeutic agents.

The Effects of Selected Sesquiterpenes from Myrica rubra Essential Oil on the Efficacy of Doxorubicin in Sensitive and Resistant Cancer Cell Lines.

β-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER) and valencene (VAL) are constituents of the essential oil of Myrica rubra (MEO), which has significant antiproliferative effect in various cancer cell lines. In the present study, we compared the antiproliferative effect of these sesquiterpenes alone and in combination with the cytostatic drug doxorubicin (DOX) in cancer cell lines with different sensitivity to DOX. Two ovarian cancer cell lines (sensitive A2780 and partly resistant SKOV3) and two lymphoblast cancer cell lines (sensitive CCRF/CEM and completely resistant CEM/ADR) were used. The observed effects varied among sesquiterpenes and also differed in individual cell lines, with only VAL being effective in all the cell lines. A strong synergism of DOX with NER was found in the A2780 cells, while DOX acted synergistically with HUM and CAO in the SKOV3 cells. In the CCRF/CEM cells, a synergism of DOX with CAO and NER was observed. In resistant CEM/ADR cells, sesquiterpenes did not increase DOX efficacy, although they significantly increased accumulation of DOX (up to 10-times) and rhodamine-123 (substrate of efflux transporter ABCB1) within cancer cells. In conclusion, the tested sesquiterpenes were able to improve DOX efficacy in the sensitive and partly resistant cancer cells, but not in cells completely resistant to DOX.

Bufalin reverses ABCB1-mediated drug resistance in colorectal cancer.

Multidrug resistance (MDR), mainly mediated by ABCB1 transporter, is a major cause for chemotherapy failure. Bufalin (BU), an active component of the traditional Chinese medicine chan’su, has been reported to have antitumor effects on various types of cancer cells. The purpose of this present study was to investigate the reversal effect of BU on ABCB1-mediated multidrug resistance in colorectal cancer. BU at safe concentration (5, 10, 20 nM) could reverse chemosensitivity of ABCB1-overexpression HCT8/ADR, LoVo/ADR and HCT8/ABCB1 nearly back to their parental cells level. In addition, results from the drug accumulation studies revealed that BU was able to enhance intracellular accumulation of doxorubicin (DOX) and Rhodamine 123 (Rho-123) in a dose-dependent manner. Furthermore, Western blot assays showed that BU significantly inhibited the expression level of ABCB1 protein. Meanwhile, BU stimulated the ATPase activity of ABCB1, which suggested that BU might be a substrate of ABCB1. More interestingly, docking analysis predicted that BU could be docked into the large hydrophobic drug-binding cavity of human ABCB1. Importantly, BU remarkable increased the effect of DOX against the ABCB1 resistant HCT8/ADR colorectal cell xenografts in nude mice, without inducing any obvious toxicity. Overall, we concluded that BU efficiently reversed ABCB1-mediated MDR through not only inhibited the efflux function of ABCB1, but also down-regulate its protein expression, which might represent a potential and superior ABCB1 modulator in colorectal cancer.

Intestinal absorption mechanisms of 2′-deoxy-2′-β-fluoro-4′-azidocytidine, a cytidine analog for AIDS treatment, and its interaction with P-glycoprotein, multidrug resistance-associated protein 2 and breast cancer resistance protein

2′-Deoxy-2′-β-fluoro-4′-azidocytidine (FNC), a cytidine analog, has attracted great interest because of its potent activity against wild-type and multidrug-resistant HIV. The purpose of current study was to investigate the absorption mechanisms of FNC in the small intestine, as well as the interactions between FNC and P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). The experiments were performed using Caco-2 cells and the rat small intestine. The uptake experiment indicated that FNC concentration, extracellular pH and the incubated temperature could influence the uptake of FNC in Caco-2 cells. NaN3, verapamil, probenecid, MK571 and GF120918 could significantly increase the FNC uptake in Caco-2 cells. The transport experiment showed that both the absorption and secretion of FNC were concentration dependent. The secretion of FNC was approximately 2-fold greater than the absorption. In the presence of verapamil, probenecid, MK571 or GF120918, the efflux ratio decreased by >50%. In everted rat intestine, the absorption of FNC also depended on its concentration and was not significantly different in the different segments of the small intestine. Real-time RT-PCR results indicated that the gene expressions of P-gp, MRP2 and BCRP were up-regulated after exposure to FNC. The reduction in accumulation of rhodamine 123 after treatment with FNC revealed its ability to up-regulate P-gp activity. In conclusion, FNC was completely absorbed by passive diffusion and active transport mechanisms. P-gp, MRP2 and BCRP could influence the absorption of FNC in the small intestine. FNC could modulate the gene expressions of P-gp, MRP2 and BCRP, and increase the activity of P-gp.

Enhanced apoptosis, survivin down-regulation and assisted immunochemotherapy by curcumin loaded amphiphilic mixed micelles for subjugating endometrial cancer

Survivin is up-regulated in 83% of endometrial cancer leading to resistance development. As endometrial tumor advances, it also elicits chronic inflammation characterized by increased cytokine secretion and immune cells infiltration. The present study was designed to engineer mixed micellar curcumin loaded formulation for investigating survivin down-regulation, its anti-cancer and cytokine modulatory potential against endometrial cancer Ishikawa cells. Flory–Huggins interaction parameter (χpd) was applied to predict the compatibility between curcumin and surfactant mixture. The developed and characterized formulations were used to comparatively assess hemolysis, cellular uptake, cell-viability, apoptosis, mitochondrial membrane potential loss, rhodamine accumulation and bioavailability. In-vitro cytotoxicity in Vero cells demonstrated no deleterious effects on cell population. We saw better bioavailability, significant rhodamine accumulation, changes in protein expression and modulation in TNF-α, IL-6 and IL-10 levels. In conclusion, developed formulation warrants exploring the therapeutic interventions for overcoming resistance development in endometrial cancer.

Hypomethylation agent decitabine restores drug sensitivity by depressing P-glycoprotein activity through MAPK signaling pathway.

The multidrug resistance (MDR) continues to be an obstacle in the treatment of both hematological and solid tumors. Hypomethylation agent, decitabine (5-Aza-dC), is an experimental agent in MDR therapy, while the mechanism is not very clear. In the present study, we demonstrated 5-Aza-dC may reverse MDR induced by P-glycoprotein (P-gp) coded by mdr1 gene in both hematologic K562/ADR cells and solid tumor MCF-7/ADR cells with time and dose-dependent manner. 5-Aza-dC significantly increased drug sensitivity in patients' leukemic cells which had higher expression of mdr1 gene. Both total protein and membrane P-gp expression were up-regulated with 5-Aza-dC treatment in K562/ADR and MCF-7/ADR cells. However, accumulation of adriamycin and rhodamine 123 were increased which suggested the depression of P-gp activity. Gene expression profiling was performed and activation of MAPK signaling pathway was identified as the most significant change affected by 5-Aza-dC. Inhibition of MAPK pathway could increase P-gp activity. Our data suggested that hypomethylation agent decitabine restores drug sensitivity in the P-gp-induced MDR phenotype by depressing of P-gp activity as drug pump partly through MAPK signaling pathway.

Cepharanthine hydrochloride reverses P‑glycoprotein-mediated multidrug resistance in human ovarian carcinoma A2780/Taxol cells by inhibiting the PI3K/Akt signaling pathway.

Ovarian cancer has the highest mortality rate among gynecologic malignant tumors. The major obstacle to treatment success is multidrug resistance (MDR) to chemotherapy drugs. Cepharanthine hydrochloride (CH), a natural alkaloid-derived compound, has shown MDR reversal potency in several tumor cell lines; however, the molecular mechanism is not entirely known. In the present study, we assessed whether CH sensitized malignant cells to chemotherapy drugs in ovarian cancer and explored the relevant mechanism. We found that CH reduced the IC50 value of paclitaxel and increased intracellular rhodamine-123 accumulation in human ovarian cancer A2780/Taxol cells in a concentration-dependent manner. Reverse transcription polymerase chain reaction and western blot assay demonstrated that CH inhibited MDR1 expression as indicated by reduced mRNA and protein levels in A2780/Taxol cells. In addition, the inhibitory effect was strengthened after CH was combined with the specific PI3K/Akt signaling pathway inhibitor LY294002. Furthermore, p‑Akt expression decreased gradually with the concentration of CH (2, 4 and 8µM). Taken together, these findings indicated that CH reversed P‑glycoprotein-mediated MDR in A2780/Taxol cells by inhibiting the PI3K/Akt signaling pathway.

Fractional laser-assisted percutaneous drug delivery via temperature-responsive liposomes

Liposomes are used for transdermal delivery of drugs and vaccines. Our objective was to develop temperature-responsive (TR) liposomes to achieve temperature-dependent, controlled release of an encapsulated drug, and use fractional laser irradiation to enhance transdermal permeability of these liposomes. TR-liposomes prepared using a thermosensitive polymer derived from poly-N-isopropylacrylamide, N,N-dimethylacrylamide, egg phosphatidylcholine, and dioleoylphosphatidylethanolamine, delivered fluorescein isothiocyanate-conjugated ovalbumin (OVA-FITC) as a model drug. Effect of temperature on liposome size and drug release rate was estimated at two temperatures. Transdermal permeation through hairless mouse skin, with and without CO2 fractional laser irradiation, and penetration into Yucatan micro-pig skin were investigated using Franz cell and fluorescence microscopy. Dynamic light scattering showed that mean liposome diameter nearly doubled from 190 to 325 nm between 37 and 50 °C. The rate and amount of OVA-FITC released from TR-liposomes were higher at 45 °C that those at 37 °C. Transdermal permeation of OVA-FITC across non-irradiated skin from both TR- and unmodified liposomes was minimal at 37 °C, but increased at 45 °C. Laser irradiation significantly increased transdermal permeation of both liposome groups at both temperatures. Fluorescence microscopy of frozen biopsy specimens showed deeper penetration of FITC from unmodified liposomes compared to that from polymer-modified liposomes. Rhodamine accumulation was not observed with polymer-modified liposomes at either temperature. Temperature-dependent controlled release of an encapsulated drug was achieved using the TR-liposomes. However, TR-liposomes showed lower skin permeability despite higher hydrophobicity. Fractional laser irradiation significantly increased the transdermal permeation. Additional studies are required to control liposome size and optimize transdermal permeation properties.

Reversal of P-gp-mediated multidrug resistance in colon cancer by cinobufagin.

Cinobufagin (CBF) is isolated from the skin and posterior auricular glands of the Asiatic toad (Bufo gargarizans). This study investigated the reversal effect of CBF on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in colon cancer. The effect of CBF on the cytotoxicity of anticancer drugs in P-gp overexpressing LoVo/ADR, HCT116/L, Cao-2/ADR cells and their parental cells was determined using CCK-8 assay. Apoptosis of anti-cancer drugs and accumulation of doxorubicin (DOX) and Rhodamine 123 (Rho123) in P-gp overexpressing cells were evaluated by flow cytometry. Results indicated that CBF significantly enhanced the sensitivity of P-gp substrate drugs on P-gp overexpressing cells, but had no effect on their parental cells. CBF enhanced the effect of DOX against P-gp-overexpressing LoVo/ADR cell xenografts in nude mice. Moreover, CBF also increased cell apoptosis of chemotherapy agents and intracellular accumulation of DOX and Rho123 in the MDR cells. Further research on the mechanisms revealed non-competitive inhibition of P-gp ATPase activity, but without altering the expression of P-gp. These findings demonstrated that CBF could be further developed into a safe and potent P-gp modulator for combination use with anticancer drugs in cancer chemotherapy.

Potent Inhibition of Nitric Oxide-Releasing Bifendate Derivatives against Drug-Resistant K562/A02 Cells in Vitro and in Vivo.

Multidrug resistance is a major obstacle to successful chemotherapy for leukemia. In this study, a series of nitric oxide (NO)-releasing bifendate derivatives (7a-n) were synthesized. Biological evaluation indicated that the most active compound (7a) produced relatively high levels of NO and significantly inhibited the proliferation of drug-resistant K562/A02 cells in vitro and in vivo. In addition, 7a induced the mitochondrial tyrosine nitration and the intracellular accumulation of rhodamine 123 by inhibiting P-gp activity in K562/A02 cells. Furthermore, 7a remarkably down-regulated AKT, NF-κB, and ERK activation and HIF-1α expression in K562/A02 cells, which are associated with the tumor cell proliferation and drug resistance. Notably, the antitumor effects were dramatically attenuated by an NO scavenger or elimination of the NO-releasing capability of 7a, indicating that NO produced by 7a contributed to, at least partly, its cytotoxicity against drug-resistant K562/A02 cells. Overall, 7a may be a potential agent against drug-resistant myelogenous leukemia.