Intracellular Accumulation Of Adriamycin Research Articles

Catalpol alleviates adriamycin-induced nephropathy by activating the SIRT1 signalling pathway in vivo and in vitro.

Catalpol, a water-soluble active ingredient isolated from Rehmannia glutinosa, exhibits multiple pharmacological activities. However, the mechanism(s) underlying protection against renal injury by catalpol remains unknown. Adriamycin-induced kidney injury models associated with podocyte damage were employed to investigate the nephroprotective effects of catalpol. In vivo, TUNEL and haematoxylin-eosin staining was used to evaluate the effect of catalpol on kidney injury in mice. In vitro, effects of catalpol on podocyte damage induced by adriamycin was determined by elisa kit, flow cytometry, Hoechst 33342, and TUNEL staining. The mechanism was investigated by siRNA, EX527, and docking simulations. In vivo, catalpol treatment significantly improved adriamycin-induced kidney pathological changes and decreased the number of apoptotic cells. In vitro, catalpol markedly decreased the intracellular accumulation of adriamycin and reduced the calcium ion level in podocytes and then attenuated apoptosis. Importantly, the regulatory effects of catalpol on sirtuin 1 (SIRT1), multidrug resistance-associated protein 2 (MRP2), and the TRPC6 channel were mostly abolished after incubation with SIRT1 siRNA or the SIRT1-specific inhibitor EX527. Furthermore, docking simulations showed that catalpol efficiently oriented itself in the active site of SIRT1, indicating a higher total binding affinity score than that of other SIRT1 activators, such as resveratrol, SRT2104, and quercetin. Taken together, our results suggest that catalpol exhibits strong protective effects against adriamycin-induced nephropathy by inducing SIRT1-mediated inhibition of TRPC6 expression and enhancing MRP2 expression.

Suppressing Hedgehog signaling reverses drug resistance of refractory acute myeloid leukemia.

BackgroundHedgehog (Hh) signaling is involved in the pathogenesis of tumors. By performing gene chip analysis, we predicted that Hh signaling might regulate multiple downstream pathways in acute myeloid leukemia (AML).MethodsIn this study, the potential role of the Hh pathway in refractory AML, and the impact of Hh expression on clinical prognosis were examined. We also investigated the role of the Hh inhibitor NVP-LDE225 in reversing drug resistance of refractory primary AML cells in vitro and the roles of multiple drug-resistant HL60/Adriamycin-resistant cells in vitro and in vivo (in a xenograft mouse model). Finally, we explored the underlying mechanisms.ResultsHh pathway was highly active in chemotherapy-resistant AML cells; by contrast, activation was less pronounced in chemosensitive cells and non-refractory primary cells. Strong activation of this pathway was associated with higher recurrence rates and poorer relapse-free and overall survival. NVP-LDE225 inhibited MRP1 protein expression, increased intracellular accumulation of Adriamycin, and reversed chemotherapeutic resistance. These effects were likely mediated through inhibition of the IGF-1R/Akt/MRP1 pathway. In the AML xenograft mouse model, NVP-LDE225 plus Adriamycin resulted in marked tumor regression.ConclusionThese findings suggest that targeting the Hh pathway might be a therapeutic avenue for overcoming MDR resistance and preventing refractory AML.

Usnea Acid as Multidrug Resistance (MDR) Reversing Agent against Human Chronic Myelogenous Leukemia K562/ADR Cells via an ROS Dependent Apoptosis.

Purpose Multidrug resistance (MDR) is a major obstacle in chemotherapy of leukemia treatments. In this paper, we investigated Usnea Acid (UA) as MDR reversal agent on hematologic K562/ADR cells via ROS dependent apoptosis. Methods CCK8 assay was used to measure cell viability rate of K562/ADR. Intracellular reactive oxygen species (ROS) generation, cell cycle distribution, cell apoptosis were measured with flow cytometry, respectively. Proteins related to apoptosis were measured by Western blot. Intracellular Adriamycin accumulation was observed by confocal microscopy and measured by flow cytometry. Results In vitro study showed intracellular Adriamycin accumulation was remarkably increased by UA. Cell viability treated with Adr (4 μM) was decreased from 89.8% ± 4.7 to 32% ± 8.9 by combined with UA (4 μM). Adr-induced apoptosis and G1/G0 phase cell cycle arrest were remarkably increased by UA, as well as, intracellular ROS level. However, MDR reversing activity of UA was inhibited by N-acetyl cysteine (NAC), a ROS scavenger. Conclusion These data provide compelling evidence that UA is a promising agent against MDR in leukemia cell line and suggest a promising therapeutic approach for leukemia.

The effects of ultrasound exposure on P-glycoprotein-mediated multidrug resistance in vitro and in vivo

BackgroundMultidrug resistance (MDR) is often responsible for the failure of chemotherapy treatment, and current strategies for cancer MDR are not adequately satisfying as to their efficacy and safety. In this study, we sought to determine the anti-MDR effects of ultrasound (US) irradiation and its underlying mechanisms against drug-resistance.MethodsMDR variant MCF-7/ADR cell lines and endothelial cell lines were used to determine the appropriate ultrasound intensity for in vitro experiments. MCF-7/ADR cell and HEPG2/ADM cells were used to assess the anti-MDR effect of US irradiation. Intracellular adriamycin (ADM) accumulation, Cell viability, cell proliferation and cell apoptosis were evaluated after ADM + US treatment or ADM treatment alone. MCF-7/ADR xenograft mice were used to investigate the appropriate ultrasound intensity for in vivo experiments and its effect on the long-term prognosis. Underlining mechanisms by which ultrasound exposure reversing MDR phenotype were investigated both in vitro and in vivo.ResultsCombination of ADM and 0.74 W/cm2 US irradiation enhanced ADM intracellular concentration and nuclear accumulation in MCF-7/ADR and HEPG2/ADM cells, compared to those treated with ADM alone. Enhanced cellular ADM uptake and nuclei localization was associated with increased cytotoxicity of ADM to ADM-resistant cells, lower ADM-resistant cell viability and proliferative cell ratio, and higher apoptotic cell ratio. More importantly, US exposure increased the effectiveness of ADM to inhibit tumor growth in MCF-7/ADR xenograft mice. Mechanistically, US exposure promoted ADM accumulation in MDR cells mainly through down-regulation of P-glycoprotein (P-gp), which is dependent on US-induced intracellular reactive oxygen species (ROS) production. US-induced oxidative stress promoted miR-200c-3p and miR-34a-3p expression by forming miR-200c/34a/ZEB1 double-negative feedback loop. Finally, US-induced miR-200c/34a overexpression decreased P-gp expression and reversed MDR phenotype.ConclusionUS irradiation could reverse MDR phenotype by activating ROS-ZEB1-miR200c/34a-P-gp signal pathway. Our findings offer a new and promising strategy for sensitizing cells to combat MDR and to improve the therapeutic index of chemotherapy.

TRIB2 knockdown as a regulator of chemotherapy resistance and proliferation via the ERK/STAT3 signaling pathway in human chronic myelogenous leukemia K562/ADM cells.

Acquired resistance to chemotherapy plays a critical role in human drug treatment failure in many tumor types. Multidrug resistance (MDR) to Adriamycin (ADM) also limits the efficacy of therapy in human chronic myelogenous leukemia (CML). The overexpression of drug efflux transporters is one mechanism uderlying MDR. In particular, the consistent activation of MDR1 and MDR‑associated protein 1 (MRP1) is involved in drug resistance. In the present study, ADM‑resistant human CML K562/ADM cells were stably transfected with a Tribbles homolog 2 (TRIB2)‑targeted vector. A CCK‑8 assay showed that the half maximal inhibitory concentration (IC50) of ADM and the cell proliferation were lower in the transfected cells compared with that in the parental K562/ADM cells. The mRNA and protein expression levels of MDR1 and MRP1 were determined by reverse transcription‑polymerase chain reaction (RT‑PCR), RT‑quantitative PCR and western blot analysis. The results showed that the expression of MDR1 and MRP1 was significantly reduced in K562/ADM cells transfected with pGPU6/GFP/Neo‑TRIB2. Due to the downregulation of MDR1 and MRP1, the intracellular accumulation of ADM was increased in the transfected cells compared with that in the parental K562/ADM cells. Therefore, the sensitivity of the K562/ADM cells to ADM was enhanced and proliferation was inhibited. Our research revealed that protein expression of the ERK signaling pathway was inhibited by downregulating TRIB2, indicating that the ERK pathway was involved in cell drug resistance and proliferation. Furthermore, we used the ERK‑specific blocker U0126 to demonstrate this phenomenon. In summary, our research suggested that knockdown of TRIB2 could slow cell growth and reverse resistance, implying that TRIB2 is a potential therapy target for resistant human CML.

Matrine reversed multidrug resistance of breast cancer MCF-7/ADR cells through PI3K/AKT signaling pathway.

Matrine is an alkaloid extracted from a Chinese herb Sophora flavescens Ait, and has been used clinically for breast cancer with marked therapeutic efficacy in China. However, the mechanism has not been well known. Thus, the present study was to explore whether Matrine reverses multidrug resistance for breast cancer cells through the regulation of PI3K/AKT signaling pathway. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the inhibitory action; Annexin V to detect apoptosis; fluorospectrophotometry to examine intracellular adriamycin (ADR) accumulation; and Western blot to label the proteins of P-glycoprotein (P-gp), MRP1, PTEN, p-AKT, Bcl-2, Bax, and Caspase-3. Matrine (0-2.5 mg/mL) inhibited MCF-7/ADR cell growth and induced apoptosis (P < 0.01). A total of 0.2 mg/mL Matrine could increase the intracellular concentration of ADR; the accumulation in MCF-7/ADR cells increased 3.56 times. Compared with control group, 0.6, 1.2 mg/mL Matrine reduced protein expressions of P-gp, MRP1, p-AKT, Bcl-2, but increased PTEN, Bax, and cleaved caspase-3 gradually, and unchanged caspase-3. Matrine was more likely to reduce the expression of P-gp, MRP1, and p-AKT at the same inhibition radio of Matrine, (0.6 mg/mL) and MK2206 (0.05 μmol/L). Matrine inhibited MCF-7/ADR cell growth, induced apoptosis, and reversed multidrug resistance for breast cancer cells through the regulation of downstream apoptosis factors of PI3K/AKT signaling pathway by decreasing cell phosphorylation of AKT level.

With no interaction, knockdown of Apollon and MDR1 reverse the multidrug resistance of human chronic myelogenous leukemia K562/ADM cells.

Chemotherapy is the main treatment method for patients with chronic myeloid leukemia(CML) and has achieved marked results. However, the acquisition of multidrug resistance(MDR) has seriously affected the quality of life and survival rate of patients. The overexpression of the inhibitors of apoptosis proteins(IAPs) and the adenosine triphosphate(ATP)-dependent binding cassette(ABC) transporters are the two main causes of MDR. Apollon and MDR1 are the most important and representative members, respectively, among the IAPs and ABC transporters. In the present study, we investigated the role of Apollon and MDR1 in chemotherapy resistance and their mechanism of interaction. We respectively knocked down the expression of Apollon and MDR1 using short hairpin RNA(shRNA) in adriamycin(ADM) resistant human CML K562 cells and examined the drug sensitivity, the consequences with regard to ADM accumulation and the alterations in the expression of Apollon and MDR1. The expression levels of Apollon and MDR1 mRNA were higher in the K562/ADM cells compared with the parental K562 cells as determined by reverse transcription‑polymerase chain reaction (RT-PCR). The plasmids of Apollon and MDR1 shRNA were respectively stably transfected into K562/ADM cells using Lipofectamine2000. The transfection efficiency was detected by fluorescence microscopy. Cell Counting Kit-8 (CCK-8) assay revealed that Apollon or MDR1 knockdown significantly increased the chemosensitivity of the K562/ADM cells to ADM. Flow cytometric assay revealed that K562/ADM/shMDR1 cells exhibited a significantly increased intracellular accumulation of ADM, and that changes were not found in the K562/ADM/shApollon cells. Compared with the parental K562/ADM cells, a significantly decreased expression of Apollon mRNA and protein was determined in the K562/ADM/shApollon cells without affecting the expression of MDR1 as determined by RT-PCR and western blotting. Likewise, the expression levels of MDR1 mRNA and protein also markedly downregulated in the K562/ADM/shMDR1 cells had no effect on Apollon expression. Collectively, our findings demonstrated, for the first time, that downregulation of Apollon or MDR1 through stable transfection with the Apollon- or MDR1-targeting shRNA induced MDR reversal through respective inhibition of Apollon or MDR1 expression and function. However, the reversal mechanism of Apollon and MDR1 revealed no direct interaction with each other.

Antiproliferative effect of ZSTK474 alone or in combination with chemotherapeutic drugs on HL60 and HL60/ADR cells.

While chemotherapy remains to be one of the main approaches in the clinical treatment of acute myeloid leukemia (AML), multidrug resistance (MDR) has become a serious problem which limits the therapeutic efficacy. The important roles of the PI3K/Akt pathway in modulating cell proliferation and MDR suggest that PI3K inhibitor might be effective for treatment of AML. In the present study, the antiproliferative effects of PI3K inhibitor ZSTK474 on AML cell HL60 and the adriamycin (ADR)-resistant HL60/ADR cells were investigated. Our data indicated that ZSTK474 exhibited potent antiproliferative activity, induced G1 cell cycle arrest, but no obvious apoptosis in both cell lines. Moreover, ZSTK474 affected the protein levels of cell-cycle-related molecules including increased p27, decreased cyclin D1 and phosphorylated Rb in dose-dependent manner. The proteins downstream of PI3K including phosphorylated PDK1, Akt and GSK-3β were reduced in a dose-dependent manner after ZSTK474 treatment. ZSTK474 reversed ADR resistance, increased the intracellular accumulation of ADR, and reduced the expression and function of multidrug resistance (MDR) proteins including both P-gp and MRP1 in HL60/ADR cells. The combination of ZSTK474 and chemotherapeutic drugs cytarabine or vincristine led to a synergistic effect in HL60 and HL60/ADR cells. In conclusion, ZSTK474 showed potent antiproliferative effect on HL60 and HL60/ADR cells; combination with cytarabine or vincristine resulted in synergistic effect. Our results suggest ZSTK474 has the potential to be applied in the treatment of AML patients, while further evidences particularly those about in vivo efficacy are needed.

Shenmai injection enhances the cytotoxicity of chemotherapeutic drugs against colorectal cancers via improving their subcellular distribution.

Shenmai injection (SMI) is a Chinese patent-protected injection, which was mainly made of Red Ginseng and Radix Ophiopogonis and widely used for treating coronary heart disease and tumors by boosting Qi and nourishing Yin. In this study we examined whether SMI could produce direct synergetic effects on the cytoxicity of adriamycin (ADR) and paclitaxel (PTX) in colorectal cancers in vivo and in vitro, and explored the underlying pharmacokinetic mechanisms. BALB/c nude mice with LoVo colon cancer xenografts were intraperitoneally injected with ADR (2 mg·kg-1·3d-1) or PTX (7.5 mg·kg-1·3d-1) with or without SMI (0.01 mL·g-1·d-1) for 13 d. Co-administration of SMI significantly enhanced the chemotherapeutic efficacy of ADR and PTX, whereas administration of SMI alone at the given dosage did not produce visible anti-cancer effects, The chemosensitizing action of SMI was associated with increased concentrations of ADR and PTX in the plasma and tumors. In Caco-2 and LoVo cells in vitro, co-treatment with SMI (2 μL/mL) significantly enhanced the cytotoxicity of ADR and PTX, and resulted in some favorable pharmacokinetic changes in the subcellular distribution of ADR and PTX. In addition, SMI-induced intracellular accumulation of ADR was closely correlated with the increased expression levels of P-glycoprotein in 4 colon cancer cell lines (r2=+0.8558). SMI enhances the anti-cancer effects of ADR and PTX in colon cancers in vivo and in vitro by improving the subcellular distributions of ADR and PTX.

Targeting autophagy augments the activity of DHA-E3 to overcome p-gp mediated multi-drug resistance.

Multidrug resistance (MDR) is a major obstacle for successful chemotherapy treatment. Searching for effective MDR modulators and combining them with anticancer drug therapies has been a promising strategy against clinical MDR. In our previous study, we have found that DHA-E3, a synthetic derivative of DHA, has the ability to modulate the function of P-glycoprotein (P-gp) and reverse MDR in cancer cells. In this study, we further evaluated the reversal effect of DHA-E3 on MDR and explored its mechanism of action in vitro. Our findings showed that DHA-E3 significantly potentiated the cytotoxicity of vincristine(VCR) and adriamycin(ADR) in the P-gp over-expressing KB/VCR and A02 cells. The mechanistic study found that DHA-E3 increased the intracellular accumulation of ADR and rhodamine-123 by directly inhibiting the drug-transport activity of P-gp. In the present study, we found that DHA-E3 not only reversed MDR, but also induced autophagy in MDR cancer cells. To determine whether DHA-E3-induced autophagy is an adaptive survival response or contributes to cell death, we manipulated autophagic activity using autophagy inhibitor 3-MA or siRNA targeting Beclin1. We found that the reversal activity of DHA-E3 was significantly exacerbated in the presence of 3-MA or blocking the expression of Beclin1. These results suggest that DHA-E3 is capable of reversing MDR, induction of autophagy represents a defense mechanism and inhibiting this process may be an effective strategy to augment the reversal activity of reversal agents.

NSCA-1-a novel N-substituted coumalamide derivative-increases Adriamycin sensitivity in HepG2/adriamycin cells through modulating Akt/GSK-3β signaling and p53-dependant apoptotic pathway.

Coumalamide derivatives are one of 2-pyrones derivatives, exerting multifunctional bioactivity. An array of coumalamide derivatives have been developed and presented good antiproliferative properties on cancer cells. However, the synthesis of 5-substituted coumalamide derivatives has not yet been published. Resistance to chemotherapeutic drugs is a major obstacle in hepatocellular carcinoma therapy. Recent evidence suggests that overexpression of constitutively active Akt confers on cancer cells resistance to chemotherapy. In this study, we report the synthesis and biological evaluation of a novel N-substituted coumalamide derivative (NSCA-1). The results indicated that NSCA-1 exerts synergistic cytotoxicity with Adriamycin in HepG2/ADR (HepG2/adriamycin) cells. Furthermore, both of the Akt kinase activity and phosphorylated Akt (Ser473) were found to be inhibited by NSCA-1 and subsequently resulting in decreased phosphorylation of GSK-3β. The intracellular accumulation of Adriamycin was also boosted by NSCA-1 via reducing the expression of p-gp. In addition, we found that combined treatment with NSCA-1 enhance cell apoptosis induced by Adriamycin via p53-dependant apoptotic pathway.

Psoralen reverses the P-glycoprotein-mediated multidrug resistance in human breast cancer MCF-7/ADR cells.

The resistance of cancer to chemotherapeutic agents is a major obstacle during chemotherapy. Clinical multidrug resistance (MDR) is commonly mediated by membrane drug efflux pumps, including ATP‑binding cassette subfamilyB member1, also termed P-glycoprotein (P-gp). P-gp is a membrane transporter encoded by the MDR1 gene. The current study aimed to investigate the impact of psoralen on the expression and function of P‑gp. The 10% inhibitory concentration (IC10) of psoralen, and its capacity to reduce MDR in adriamycin (ADR)‑resistant MCF‑7/ADR cells were determined using MTT assay. The ability of psoralen to modulate the transport activity of P‑gp in MCF‑7/ADR cells was evaluated by measuring the accumulation and efflux of rhodamine123 (Rh123) and adriamycin with flow cytometry. The present study evaluated the mRNA level of MDR1 in MCF‑7and MCF‑7/ADR cells treated with psoralen using reverse transcription-quantitative polymerase chain reaction. The protein expression level of P‑gp was examined by western blot analysis. The current study demonstrated that the IC10 of psoralen in MCF‑7/ADR cells was 8µg/ml. At 8µg/ml, psoralen reduced MDR and the sensitivity of the MCF‑7/ADR cells to ADR compared with untreated cells. Additionally, psoralen significantly increased the intracellular accumulation of ADR and Rh123. However, the IC10 of psoralen did not affect the protein expression levels of P‑gp or mRNA levels of MDR1 (P>0.05). Psoralen reduces MDR by inhibiting the efflux function of P‑gp, which may be important for increasing the efficiency of chemotherapy and improving the clinical protocols aiming to reverse P-gp-mediated MDR.

Design, synthesis and biological evaluation of LBM-A5 derivatives as potent P-glycoprotein-mediated multidrug resistance inhibitors

A novel series of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) inhibitors with triazol-N-phenethyl-tetrahydroisoquinoline or triazol-N-ethyl-tetrahydroisoquinoline scaffold were designed and synthesized via click chemistry. Most of the synthesized compounds showed higher reversal activity than verapamil (VRP). Among them, the most potent compound 4 showed a comparable activity with the known potent P-gp inhibitor WK-X-34 with lower cytotoxicity toward K562 cells (IC50>100μM). Compared with VRP, compound 4 exhibited more potency in increasing drug accumulation in K562/A02 MDR cells. Moreover, compound 4 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 4 could remarkably increase the intracellular accumulation of Adriamycin (ADM) in K562/A02 cells as well as inhibit rhodamine-123 (Rh123) efflux from the cells. These results suggested that compound 4 may represent a promising candidate for developing P-gp-mediated MDR inhibitors.

Chaetominine reduces MRP1-mediated drug resistance via inhibiting PI3K/Akt/Nrf2 signaling pathway in K562/Adr human leukemia cells

Drug resistance limits leukemia treatment and chaetominine, a cytotoxic alkaloid that promotes apoptosis in a K562 human leukemia cell line via the mitochondrial pathway was studied with respect to chemoresistance in a K562/Adr human resistant leukemia cell line. Cytotoxicity assays indicated that K562/Adr resistance to adriamycin (ADR) did not occur in the presence of chaetominine and that chaetominine increased chemosensitivity of K562/Adr to ADR. Data show that chaetominine enhanced ADR-induced apoptosis and intracellular ADR accumulation in K562/Adr cells. Accordingly, chaetominine induced apoptosis by upregulating ROS, pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2. RT-PCR and western-blot confirmed that chaetominine suppressed highly expressed MRP1 at mRNA and protein levels. But little obvious alternation of another drug transporter MDR1 mRNA was observed. Furthermore, inhibition of MRP1 by chaetominine relied on inhibiting Akt phosphorylation and nuclear Nrf2. In summary, chaetominine strongly reverses drug resistance by interfering with the PI3K/Akt/Nrf2 signaling, resulting in reduction of MRP1-mediated drug efflux and induction of Bax/Bcl-2-dependent apoptosis in an ADR-resistant K562/Adr leukemia cell line.

Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML.

Timosaponin A-III reverses multi-drug resistance in human chronic myelogenous leukemia K562/ADM cells via downregulation of MDR1 and MRP1 expression by inhibiting PI3K/Akt signaling pathway.

One of the major causes of failure in chemotherapy for patients with human chronic myelogenous leukemia (CML) is the acquisition of multidrug resistance (MDR). MDR is often associated with the overexpression of drug efflux transporters of the ATP-binding cassette (ABC) protein family. Timosaponin A-III (TAIII), a saponin isolated from the rhizome of Anemarrhena asphodeloides, has previously demonstrated the ability to suppress certain human tumor processes and the potential to be developed as an anticancer agent. Nevertheless, the ability of TAIII to reverse MDR has not yet been explored. In this study, the adriamycin (ADM) resistance reversal effect of TAIII in human CML K562/ADM cells and the underlying mechanism was investigated. The Cell Counting Kit-8 (CCK-8) assay showed that TAIII had a reversal effect on the drug resistance of K562/ADM cells. Flow cytometry assay showed increased intracellular accumulation of ADM after cells were pretreated with TAIII, and the changes in the accumulation of rhodamine-123 (Rho-123) and 5(6)-carboxyfluorescein diacetate (CFDA) dye in K562/ADM cells were determined to be similar to the changes of intracellular accumulation of ADM. After pretreatment of cells with TAIII, the decreasing expression of P-gp and MRP1 mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). Western blotting showed TAIII inhibiting P-gp and MRP1 expression depended on the PI3K/Akt signaling pathway by decreasing the activity of p-Akt. Moreover, wortmannin an inhibitor of PI3K/Akt signaling pathway has a strong inhibitory effect on the expression of p-Akt, P-gp and MRP1. Besides, the combined treatment with TAIII did not have an affect on wortmannin downregulation of p-Akt, P-gp and MRP1. Taken together, our findings demonstrate, for the first time, that TAIII induced MDR reversal through inhibition of P-gp and MRP1 expression and function with regained adriamycin sensitivity which might mainly correlate to the regulation of PI3K/Akt signaling pathway.

A 20(S)-protopanoxadiol derivative overcomes multi-drug resistance by antagonizing ATP-binding cassette subfamily B member 1 transporter function.

In cancer cells, failure of chemotherapy is often caused by the ATP-binding cassette subfamily B member 1 (ABCB1), and few drugs have been successfully developed to overcome ABCB1-mediated multi-drug resistance (MDR). To suppress ABCB1 activity, we previously designed and synthesized a new series of derivatives based on 20(S)-protopanoxadiol (PPD). In the present study, we investigated the role of PPD derivatives in the function of ABC transporters. Non-toxic concentrations of the PPD derivative PPD12 sensitized ABCB1-overexpressing cells to their anti-cancer substrates better than either the parental PPD or inactive PPD11. PPD12 increased intracellular accumulation of adriamycin and rhodamine123 in resistant cancer cells. Although PPD12 did not suppress the expression of ABCB1 mRNA or protein, it stimulated the activity of ABCB1 ATPase. Because PPD12 is a competitive inhibitor, it was predicted to bind to the large hydrophobic cavity of homology-modeled human ABCB1. PPD12 also enhanced the efficacy of adriamycin against ABCB1-overexpressing KB/VCR xenografts in nude mice. In conclusion, PPD12 enhances the efficacy of substrate drugs in ABCB1-overexpressing cancer cells. These findings suggest that a combination therapy consisting of PPD12 with conventional chemotherapeutic agents may be an effective treatment for ABCB1-mediated MDR cancer patients.

Hh/IGF-1R/PI3K/Akt/MRP1 Pathway Induce Refractory Acute Myeloid Leukemia and Its Targeting Therpy

Hh/IGF-1R/PI3K/Akt/MRP1 pathway induce refractory acute myeloid leukemia Background: Although a great progress has made in the treatment of acute myeloid leukemia(AML), chemoresistance and relapse due to multidrug resistance (MDR) remains a therapeutic challenge. We have proven that LBH589 combined with Bortezomib had synergistic effects on proliferation, apoptosis and sensitivity to cytotoxic drugs via AKT and NF-κB pathways in chemoresistant HL60/ADR cells and refractory AML primary cells. Using gene chip analysis, we found that among those disregulated pathways, Hedgehog(Hh) signaling pathway was probably the upstream paths which could regulate others.Aims: The purpose of the study was to investigate the expression of Hh pathway in AML including primary cells and cell lines, analyze the relationship between Hh expression and clinical prognosis. Also investigate the biological effect of Hh pathway inhibitor NVP-LDE225 in reversing drug-resistance in HL60/adriamycin-resistant(ADR) cells and refractory AML primary cells and the underlying mechanisms. Besides, animal experiments were done to verify the results in vitro.Methods: Western blot assay were used to determine the protein expression of Ptch–Smo–Gli-1–Shh in HL60 and HL60/ADR cell lines and refractory or non-refractory primary AML cells. Kaplan–Meier curves were used to estimate Relapse-free survival(RFS) and Over-all survival(OS). HL60/ADR cells and refractory primary cells were treated with adriamycin or combined with NVP-LDE225 concentration among 0-10µM). Proliferation were evaluated by 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assay and cell apoptosis were analyzed by Annexin V-FITC/PI staining through flow-cytometry(FCM). Intercellular adriamycin accumulation(MFI) were analyzed by FCM. The changes in protein levels of Gli-1–IGF-1R–p-IGF-1R–IRS-1–Akt–p-Akt–MRP-1–Bcl-2 were detected by Western blot. Besides, we used a nude mouse xenograft model to verify the anti-proliferative effects in vivo.Results: We found that the refractory primary AML cells and HL60/ADR cells correlated with higher activation of the Hh pathway, however in non-refractory primary cells or chemosensitive cell lines HL60, such activation was less pronounced. Higher protein expression of this pathway was related to higher recurrence rate and associated with poor relapse free survival (RFS) and poor overall survival (OS)(P=0.002)(a). NVP-LDE225, a potent and selective Hh inhibitor, significantly reverted resistance of adriamycin(reversal fold was 3.75), induced cell apoptosis(P=0.007), increased the intracellular adriamycin accumulation(P=0.000), decreased protein expression of p-IGF-1R, IRS-1, p-Akt, Bcl-2, MRP1 also Gli-1(b). These effects were likely to be mediated via inhibition of IGF-1R/PI3K/Akt/MRP1 pathway. Besides, in a AML xenograft mouse model, NVP-LDE225 combined with ADM resulted in significant tumor regression(P=0.002) (c).Conclusions: These findings provided evidence that targeting the Hh pathway might be a therapeutic avenue for overcoming MDR resistance in myeloid leukemia.Keywords: Hedgehog signaling, acute myeloid leukemia, IGF-1R/PI3K/Akt signaling DisclosuresNo relevant conflicts of interest to declare.

Design, synthesis and evaluation of novel triazole core based P-glycoprotein-mediated multidrug resistance reversal agents

A novel series of triazol-N-ethyl-tetrahydroisoquinoline based compounds were designed and synthesized via click chemistry. Most of the synthesized compounds showed P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) reversal activities. Among them, compound 7 with little cytotoxicity towards GES-1 cells (IC50 >80μM) and K562/A02 cells (IC50 >80μM) exhibited more potency than verapamil (VRP) on increasing anticancer drug accumulation in K562/A02 cells. Moreover, compound 7 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 7 in reversing MDR revealed that it could remarkably increase the intracellular accumulation of both rhodamine-123 (Rh123) and adriamycin (ADM) in K562/A02 cells as well as inhibit their efflux from the cells. These results suggested that compound 7 showed more potency than the classical P-gp inhibitor VRP under the same conditions, which may be a promising P-gp-mediated MDR modulator for further development.

Reversing Multidrug-Resistant by RNA Interference Through Silencing MDR1 Gene in Human Hepatocellular Carcinoma Cells Subline Bel-7402/ADM

Multidrug resistance (MDR) in hepatocellular carcinoma (HC) significantly impedes the effect of chemotherapy and is considered as a primary reason leading to its recurrences and metastasis. The aim of present study was to explore new molecular targets for the reversal of MDR in HC by screening the adriamycin (ADM)-induced, human MDR-resistant HC cell subline Bel-7402/ADM. Small interfering RNAs (siRNAs) of four (MDR1si326, MDR1si1513, MDR1si2631 and MDR1si3071) targeting MDR1 were designed and transfected into Bel-7402/ADM cell strains. The experiments involved the following: mRNA expression of MDR1 gene by RT-PCR, P-glycoprotein (P-gp) expression by Western blot, intracellular ADM accumulation flow cytometry, and IC50 of ADM by a cytotoxic MTT assay. Four siRNAs reversed MDR in HC mediated by MDR1 to varying degrees. The expression level of MDR1 mRNA in cells of MDR1si326 or MDR1si2631 group (0.190 ± 0.038 or 0.171 ± 0.011) was more decreased. The expression level of P-gp in cells of MDR1si326 group was the lowest. The accumulation of ADM in cells of MDR1si326 or MDR1si2631 group (77.0 ± 3.5 or 75.4 ± 2.9) was more increased. The IC50 of cells to ADM was lowest in MDR1si326 group (11.32 ± 0.69 mg/L). Compared with other three siRNAs, MDR1si326 performed the optimal reversal effect of drug resistance in human HC Bel-7402/ADM.