Multiple Drug Resistance In Cancer Research Articles

Membrane-Fusing Vehicles for Re-Sensitizing Transporter-Mediated Multiple-Drug Resistance in Cancer.

Reversing the multiple drug resistance (MDR) arising from the overexpression of the efflux transporters often fails mainly due to the high toxicity or the poor water solubility of the inhibitors of these transporters. Here, we demonstrate the delivery of an inhibitor targeting three ABC transporters (ABCB1, ABCC1 and ABCG2) directly to the cell membrane using membrane-fusing vehicles (MFVs). Three different transfected MDCK II cell lines, along with parental cells, were used to investigate the inhibitory effect of cyclosporine A (CsA) in solution versus direct delivery to the cell membrane. CsA-loaded MFVs successfully reversed MDR for all three investigated efflux transporters at significantly lower concentrations compared with CsA in solution. Results showed a 15-fold decrease in the IC50 value for ABCB1, a 7-fold decrease for ABCC1 and an 11-fold decrease for ABCG2. We observed binding site specificity for ABCB1 and ABCG2 transporters. Lower concentrations of empty MFVs along with CsA contribute to the inhibition of Hoechst 33342 efflux. However, higher concentrations of CsA along with the high amount of MFVs activated transport via the H-binding site. This supports the conclusion that MFVs can be useful beyond their role as delivery systems and also help to elucidate differences between these transporters and their binding sites.

Abstract 1864: New generation of fully human anti-progranulin monoclonal antibodies inhibiting migration and tumor formation of non-small cell carcinoma cells

Abstract Advanced carcinomas of the lung or the breast are characterized by invasiveness and poor survival. Identifying novel targeted therapies to potentiate standard of care (SOC) therapy, remains an unmet need. In the past 4 years, numerous targeted therapies in Oncology have been approved by the Food and Drug Administration (FDA). However, the identification of targeted therapies against direct tumor biological drivers remains important. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in multiple cancers including breast and lung cancers. PGRN/GP88 tissue expression is an independent prognostic factor of recurrence and mortality while elevated serum PGRN/GP88 level is associated with poor outcomes such as progression of disease and shortened survival. The importance of inhibiting PGRN/GP88 expression or action effect on the proliferation and tumor growth of several cancers has been established by several laboratories including ours. In the current study, in association with Precision Antibody, we developed anti-PGRN/GP88 fully human monoclonal antibodies by immunizing TC transgenic humanized mice with recombinant human progranulin. Within 60 days following the start of immunization, several fully human anti-progranulin monoclonal antibodies were developed, characterized by several functional assays including neutralization, inhibition of PGRN/GP88 cell surface binding, proliferation, migration in vitro and tumor formation in vivo. Results: The inhibition of PGRN/GP88 action by fully human monoclonal antibodies inhibited cell proliferation and migration in a dose-and time-dependent fashion. Octet analysis determined Kd in the range of 10−11 M to 10−10 M. Transwell assay showed that anti-PGRN treatment inhibited migration of non-small cell lung cancer cells such as H1299 and A549 cells. In vivo xenograft studies with H1299 cells injected in athymic nude mice showed that fully human anti-PGRN antibodies inhibited tumor growth when compared to antibody control treated mice. Conclusion: PGRN/GP88 represents a therapeutic target for non-small cell carcinoma with two companion diagnostics (tissue test and ELISA to measure GP88 circulating levels). The use of the TC transgenic mice allows for the rapid development of fully human high affinity monoclonal antibodies bypassing the need for chimerization or humanization and affinity maturation of monoclonal antibodies raised in mice or other humanized systems. This work is supported by SBIR grants R44 CA 224718 and R44CA162729 from the National Cancer Institute to GS. Citation Format: Ginette Serrero, Jianping Dong, Binbin Yue, Mitsuo Oshimura, Chun Dong, Jun Hayashi. New generation of fully human anti-progranulin monoclonal antibodies inhibiting migration and tumor formation of non-small cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1864.

TAOK3 Facilitates Esophageal Squamous Cell Carcinoma Progression and Cisplatin Resistance Through Augmenting Autophagy Mediated by IRGM.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers because of its robust aggressive phenotype and chemoresistance. TAO kinase belongs to mitogen-activated protein kinases, which mediate drug resistance in multiple cancers. However, the role of TAO kinase in ESCC progression and chemoresistance has never been explored. Here, it is reported that TAOK3 augments cell autophagy and further promotes ESCC progression and chemoresistance. Mechanistically, TAOK3 phosphorylates KMT2C at S4588 and strengthens the interaction between KMT2C and ETV5. Consequently, the nuclear translocation of KMT2C is increased, and the transcription of autophagy-relevant gene IRGM is further upregulated. Additionally, the inhibitor SBI-581 can significantly suppress cell autophagy mediated by TAOK3 and synergizes with cisplatin to treat ESCC in vitro and in vivo.

Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A.

Drug resistance is a major problem associated with anticancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms have revealed that enzymes of the APOBEC3 (A3) family contribute to the development of drug resistance in multiple cancers. A3 enzymes are polynucleotide cytidine deaminases that convert cytosine to uracil (C→U) in single-stranded DNA (ssDNA) and in this way protect humans against viruses and mobile retroelements. On the other hand, cancer cells use A3s, especially A3A and A3B, to mutate human DNA, and thus by increasing rates of evolution, cancer cells escape adaptive immune responses and resist drugs. However, as A3A and A3B are non-essential for primary metabolism, their inhibition opens up a strategy to augment existing anticancer therapies and suppress cancer evolution. To test our hypothesis that pre-shaped ssDNA mimicking the U-shape observed in ssDNA-A3 complexes can provide a better binder to A3 enzymes, a Cu(I)-catalyzed azide-alkyne cycloaddition was used to cross-link two distant modified nucleobases in ssDNA. The resultant cytosine-containing substrate, where the cytosine sits at the apex of the loop, was deaminated faster by the engineered C-terminal domain of A3B than a standard, linear substrate. The cross-linked ssDNA was converted into an A3 inhibitor by replacing the 2'-deoxycytidine in the preferred TCA substrate motif by 2'-deoxyzebularine, a known inhibitor of single nucleoside cytidine deaminases. This strategy yielded the first nanomolar inhibitor of engineered A3BCTD and wild-type A3A (Ki = 690 ± 140 and 360 ± 120 nM, respectively), providing a platform for further development of powerful A3 inhibitors.

CSE1L promotes nuclear accumulation of transcriptional coactivator TAZ and enhances invasiveness of human cancer cells

The transcriptional coactivator with PDZ-binding motif (TAZ) (WWTR1) induces epithelial–mesenchymal transition and enhances drug resistance in multiple cancers. TAZ has been shown to interact with transcription factors in the nucleus, but when phosphorylated, translocates to the cytoplasm and is degraded through proteasomes. Here, we identified a compound TAZ inhibitor 4 (TI-4) that shifted TAZ localization to the cytoplasm independently of its phosphorylation. We used affinity beads to ascertain a putative target of TI-4, chromosomal segregation 1 like (CSE1L), which is known to be involved in the recycling of importin α and as a biomarker of cancer malignancy. We found that TI-4 suppressed TAZ-mediated transcription in a CSE1L-dependent manner. CSE1L overexpression increased nuclear levels of TAZ, whereas CSE1L silencing delayed its nuclear import. We also found via the in vitro coimmunoprecipitation experiments that TI-4 strengthened the interaction between CSE1L and importin α5 and blocked the binding of importin α5 to TAZ. WWTR1 silencing attenuated CSE1L-promoted colony formation, motility, and invasiveness of human lung cancer and glioblastoma cells. Conversely, CSE1L silencing blocked TAZ-promoted colony formation, motility, and invasiveness in human lung cancer and glioblastoma cells. In human cancer tissues, the expression level of CSE1L was found to correlate with nuclear levels of TAZ. These findings support that CSE1L promotes the nuclear accumulation of TAZ and enhances malignancy in cancer cells.

Molecular associations and clinical significance of core NHEJ pathway genes in renal clear cell carcinoma

Non-homologous End Joining (NHEJ) DNA damage repair pathway is the predominant pathway of DNA double-strand repair in human cells and its aberrant activity is associated with drug resistance in multiple cancers. However, the role of the NHEJ pathway in clear cell type of renal cell carcinoma (ccRCC) has been rarely addressed. We utilized authoritative databases of ccRCC containing multi-omics data, such as mutation, gene expression, DNA methylation, copy number alterations, and high throughput protein estimations to determine alterations in seven core NHEJ genes, including XRCC4, XRCC5, XRCC6, PRKDC, LIG4, NHEJ1, and PAXX. We further determined the association of their expression with clinicopathological features and molecular alterations in ccRCC. A comprehensive analysis of these genes revealed several alterations in the expression of these genes. Interestingly, NHEJ1 is downregulated and associated with better prognosis while PAXX is upregulated in ccRCC and associated with poor prognosis. These results suggest that alterations in DNA repair pathway are common in ccRCC and may provide novel therapeutic opportunities for this malignancy.

FEN1 is a prognostic biomarker for ER+ breast cancer and associated with tamoxifen resistance through the ERα/cyclin D1/Rb axis.

BackgroundTamoxifen is an important choice in endocrine therapy for patients with oestrogen receptor-positive (ER+) breast cancer, and disease progression-associated resistance to tamoxifen therapy is still challenging. Flap endonuclease-1 (FEN1) is used as a prognostic biomarker and is considered to participate in proliferation, migration, and drug resistance in multiple cancers, especially breast cancer, but the prognostic function of FEN1 in ER+ breast cancer, and whether FEN1 is related to tamoxifen resistance or not, remain to be explored.MethodsOn-line database Kaplan-Meier (KM) plotter, GEO datasets, and immunohistochemistry were used to analyse the prognostic value of FEN1 in ER+ breast cancer from mRNA and protein levels. Cell viability assay and colony formation assays showed the response of tamoxifen in MCF-7 and T47D cells. Microarray data with FEN1 siRNA versus control group in MCF-7 cells were analysed by Gene Set Enrichment Analysis (GSEA). The protein levels downstream of FEN1 were detected by western blot assay.ResultsER+ breast cancer patients who received tamoxifen for adjuvant endocrine therapy with poor prognosis showed a high expression of FEN1. MCF-7 and T47D appeared resistant to tamoxifen after FEN1 over-expression and increased sensitivity to tamoxifen after FEN1 knockdown. Importantly, FEN1 over-expression could activate tamoxifen resistance through the ERα/cyclin D1/Rb axis.ConclusionsAs a biomarker of tamoxifen effectiveness, FEN1 participates in tamoxifen resistance through ERα/cyclin D1/Rb axis. In the future, reversing tamoxifen resistance by knocking-down FEN1 or by way of action as a small molecular inhibitor of FEN1 warrants further investigation.

Silencing of the chemokine CXC receptor 4 (CXCR4) hampers cancer progression and increases cisplatin (DDP)-sensitivity in clear cell renal cell carcinoma (ccRCC)

ABSTRACT Aberrant expression of the chemokine CXC receptor 4 (CXCR4) is closely associated with cancer progression and drug-resistance in multiple cancers, and we first investigated the role of CXCR4 in regulating cancer pathogenesis and cisplatin (DDP)-resistance in clear cell renal cell carcinoma (ccRCC) in the present study. Here, we identified that CXCR4 acted as an oncogene to promote cancer progression and genetically silencing of CXCR4 increased cisplatin (DDP)-sensitivity in ccRCC in vitro and in vivo. Functionally, analysis from the clinical and cellular data indicated that CXCR4 was significantly upregulated in ccRCC tissues and cells, compared to their normal counterparts. Next, the loss-of-function experiments validated that knock-down of CXCR4 suppressed cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) in ccRCC cells, while CXCR4 overexpression had opposite effects on the above cellular functions. Consistently, the xenograft tumor-bearing mice models were established, and the results supported that knock-down of CXCR4 inhibited tumor growth and the expression levels of Ki67 protein in vivo. In addition, the ccRCC cells were exposed to DDP treatment, and we surprisingly found that upregulation of CXCR4 increased DDP-resistance in ccRCC cells, and conversely, CXCR4 ablation sensitized ccRCC cells to DDP stimulation. Taken together, we concluded that CXCR4 ablation hindered cancer progression and enhanced DDP-sensitivity in ccRCC, and the present study identified a novel therapeutic biomarker for ccRCC.

Decreased drug resistance of bladder cancer using phytochemicals treatment.

The aim of the study is to investigate the ability of phytochemicals to overcome the multiple drug resistance (MDR) of bladder cancer. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic sensitivity of T24-GCB cells, a GCB resistant cell line, to different phytochemicals, including capsaicin, quercetin, curcumin, and resveratrol, and their combination with gemcitabine. Western blot analysis was used to detect the expression of membranous ABCC2 and metabolic proteins, DCK, TK1, and TK2 in tumor cells. Animal models were used to confirm the treatment efficacy of phytochemicals in combination with gemcitabine to bladder cancer. The observed/expected ratio of cytotoxicity analysis revealed that capsaicin has synergistic effect with gemcitabine to T24-GCB cells in a dose-dependent pattern. Quercetin, curcumin, and resveratrol have additive effect with gemcitabine to T24-GCB cells. Capsaicin and quercetin alone and combination with gemcitabine decreased the expression of ABCC2 and DCK and TKs, in T24-GCB cells. On the contrary, resveratrol and curcumin alone and combination with gemcitabine increased the expression of ABCC2 but decreased cytoplasmic kinases simultaneously. In xenografted subcutaneous tumor model on nude mice, combination treatment of capsaicin and gemcitabine demonstrated the highest tumor suppression effect when compared to capsaicin or gemcitabine treatment alone. The MDR of bladder cancer is closely related to membranous ABCC2, cytoplasmic DCK, and TKs expression. Capsaicin owns the strongest synergistic cytotoxic effect of gemcitabine to T24-GCB cells. This combination regimen may provide as an adjunctive treatment for overcoming MDR in bladder cancer.

Lnc-PICSAR contributes to cisplatin resistance by miR-485-5p/REV3L axis in cutaneous squamous cell carcinoma.

BackgroundDysregulation of long noncoding RNAs (lncRNAs) is associated with drug resistance in multiple cancers. We explored the roles of lncRNA p38 inhibited cutaneous squamous cell carcinoma-associated lincRNA (PICSAR) in cisplatin (DDP) resistance of cutaneous squamous cell carcinoma (CSCC).MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure the expression of lnc-PICSAR, miR-485-5p and reversionless 3-like (REV3L) mRNA. The cell counting kit-8 (CCK-8) assay was conducted to evaluate DDP resistance and cell viability. The transwell assay was performed to determine cell migration and invasion. Western blot assay and immunohistochemistry (IHC) staining assay were carried out to measure protein levels. The dual-luciferase reporter assay was used to investigate the association between miR-485-5p and lnc-PICSAR or REV3L. Murine xenograft model was constructed to explore the function of lnc-PICSAR in vivo. The morphology of exosomes was analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA).ResultsLnc-PICSAR was elevated in DDP-resistant CSCC cells. Lnc-PICSAR silencing suppressed cell viability, DDP resistance, migration and invasion in DDP-resistant CSCC cells. MiR-485-5p acted as a target of lnc-PICSAR, and miR-485-5p inhibition reversed the impacts of lnc-PICSAR silencing on DDP resistance and cell progression in DDP-resistant CSCC cells. Lnc-PICSAR promoted REV3L expression via sponging miR-485-5p. Moreover, REV3L overexpression overturned the effects of lnc-PICSAR on cell progression and DDP resistance. Lnc-PICSAR knockdown suppressed DDP resistance in vivo. In addition, lnc-PICSAR was increased in the exosomes derived from CSCC patients’ serum and CSCC cells.ConclusionLnc-PICSAR enhanced DDP resistance via miR-485-5p/REV3L axis in DDP-resistant CSCC cells. Besides, exosome-mediated lnc-PICSAR might be involved in the regulation of drug resistance in CSCC.

Overexpression of RPN2 suppresses radiosensitivity of glioma cells by activating STAT3 signal transduction

BackgroundRadiation therapy is the primary method of treatment for glioblastoma (GBM). Therefore, the suppression of radioresistance in GBM cells is of enormous significance. Ribophorin II (RPN2), a protein component of an N-oligosaccharyl transferase complex, has been associated with chemotherapy drug resistance in multiple cancers, including GBM. However, it remains unclear whether this also plays a role in radiation therapy resistance in GBM.MethodsWe conducted a bioinformatic analysis of RPN2 expression using the UCSC Cancer Genomics Browser and GEPIA database and performed an immunohistochemical assessment of RPN2 expression in biopsy specimens from 34 GBM patients who had received radiation-based therapy. We also studied the expression and function of RPN2 in radiation-resistant GBM cells.ResultsWe found that RPN2 expression was upregulated in GBM tumors and correlated with poor survival. The expression of RPN2 was also higher in GBM patients with tumor recurrence, who were classified to be resistant to radiation therapy. In the radiation-resistant GBM cells, the expression of RPN2 was also higher than in the parental cells. Depletion of RPN2 in resistant cells can sensitize these cells to radiation-induced apoptosis, and overexpression of RPN2 had the reverse effect. Myeloid cell leukemia 1 (MCL1) was found to be the downstream target of RPN2, and contributed to radiation resistance in GBM cells. Furthermore, STAT3 was found to be the regulator of MCL1, which can be activated by RPN2 dysregulation.ConclusionOur study has revealed a novel function of RPN2 in radiation-resistant GBM, and has shown that MCL1 depletion or suppression could be a promising method of therapy to overcome the resistance promoted by RPN2 dysregulation.

NEK2 induces autophagy-mediated bortezomib resistance by stabilizing Beclin-1 in multiple myeloma.

NEK2 is associated with drug resistance in multiple cancers. Our previous studies indicated that high NEK2 confers inferior survival in multiple myeloma (MM); thus, a better understanding of the mechanisms by which NEK2 induces drug resistance in MM is required. In this study, we discovered that NEK2 enhances MM cell autophagy, and a combination of autophagy inhibitor chloroquine (CQ) and chemotherapeutic bortezomib (BTZ) significantly prevents NEK2‐induced drug resistance in MM cells. Interestingly, NEK2 was found to bind and stabilize Beclin‐1 protein but did not affect its mRNA expression and phosphorylation. Moreover, autophagy enhanced by NEK2 was significantly prevented by knockdown of Beclin‐1 in MM cells, suggesting that Beclin‐1 mediates NEK2‐induced autophagy. Further studies demonstrated that Beclin‐1 ubiquitination is decreased through NEK2 interaction with USP7. Importantly, knockdown of Beclin‐1 sensitized NEK2‐overexpressing MM cells to BTZ in vitro and in vivo. In conclusion, we identify a novel mechanism whereby autophagy is activated by the complex of NEK2/USP7/Beclin‐1 in MM cells. Targeting the autophagy signaling pathway may provide a promising therapeutic strategy to overcome NEK2‐induced drug resistance in MM.

DJ-1 overexpression confers the multidrug resistance phenotype to SGC7901 cells by upregulating P-gp and Bcl-2

Gastric cancer (GC) is one of the most malignant tumors with high incidence and mortality worldwide, and the multidrug resistance (MDR) often results in chemotherapy failure in GC. DJ-1 has been well indicated to be associated with drug resistance in multiple cancers. However, the role of DJ-1 in the MDR of gastric cancer cells and its possible mechanism remain to be elucidated. Therefore, the current study was investigated whether DJ-1 expression is differential in parental gastric cancer cell SGC7901 and vincristine (VCR)-induced gastric cancer MDR cell SGC7901/VCR, and whether DJ-1 plays a significant role in development of MDR in gastric cancer. The results showed that DJ-1 expression in SGC7901/VCR cells was significantly higher than its sensitive parental SGC7901 cells. Furthermore, DJ-1 overexpressed gastric cancer cell line SGC7901/LV-DJ-1 led to the increase of cell survival rate, the IC50 of chemotherapeutic drugs and number of cell clones as well as decrease of cell cycle G0/G1 phase ratio compared with its parental cells under the treatment of VCR, adriamycin (ADR), 5-Fluorouracil (5-FU) and cisplatin (DDP). However, the DJ-1 knockdown stable cell line SGC7901/VCR/shDJ-1 reversed the above mentioned series of MDR. Moreover, it was found that upregulation of DJ-1 protein expression promoted the pumping rate of GC cells to ADR and reduced the apoptotic index of GC cells treated with chemotherapeutic drugs by upregulating P-gp and Bcl-2. Similarly, knocking down DJ-1, P-gp or Bcl-2 displayed a converse effect. In conclusion, the current study demonstrated that DJ-1 overexpression confers the MDR phenotype to SGC7901 cells and this process is related to DJ-1 promoting active efflux of drugs and enhancing the anti-apoptotic ability of MDR GC cells by upregulating P-gp and Bcl-2.

Environmentally responsive dual-targeting nanotheranostics for overcoming cancer multidrug resistance

The development of multiple drug resistance (MDR) to chemotherapy and subsequent treatment failures are major obstacles in cancer therapy. An attractive option for combating MDR is inhibiting the expression of P-glycoprotein (P-gp) in tumor cells. Here, we report a novel chemosensitizing agent, XMD8-92, which can down-regulate P-gp. To enhance the specificity of MDR chemotherapy, a promising nanotheranostic micelle system based on poly(ethylene glycol)-blocked-poly(L-leucine) (PEG-b-Leu) was developed to simultaneously carry the anticancer drug doxorubicin, chemosensitizing agent XMD8-92, and superparamagnetic iron oxide nanoparticles (SPIOs). Featured with MDR environmentally responsive dual-targeting capability, controllable drug delivery, and efficient magnetic resonance (MR) imaging characteristics, the prepared nanotheranostics (DXS@NPs) showed outstanding in vitro cytotoxicity on MDR cells (SCG 7901/VCR) with only 53% of cells surviving compared to 90% of DOX-treated cells. Furthermore, efficient tumor inhibition and highly reduced systemic toxicity were exhibited by MDR tumor-bearing mice treated with DXS@NPs. Overall, the environmentally responsive dual-targeting nanotheranostics represent a promising approach for overcoming cancer MDR.

MiR-3619-5p hampers proliferation and cisplatin resistance in cutaneous squamous-cell carcinoma via KPNA4

Cutaneous squamous cell carcinoma (CSCC) remains the second most prevailing cancer worldwide and presents high mortality rates. Given that chemoresistance becomes an enormous obstacle to the therapy for CSCC patients, there is a pressing need to discover novel strategies for enhancing the response of CSCC cells to cisplatin. Emerging evidence has unfolded that miRNAs are participated in regulation of drug resistance in multiple cancers. MiR-3619-5p has been proofed to exert tumor inhibitive activities in human malignancies, but the biological function of miR-3619-5p in the progression of CSCC is still unclear. In this study, we observed that miR-3619-5p expression was pronouncedly dropped in cisplatin-resistant CSCC cells. Subsequently, miR-3619-5p was validated to act as a tumor suppressor in CSCC through retarding cell proliferation and cisplatin resistance. Besides, our findings certified that KPNA4 was highly expressed in cisplatin-resistant CSCC cells. Further, KPNA4 was negatively regulated by miR-3619-5p. Rescue experiments unveiled that KPNA4 counteracted the miR-3619-5p-mediated regulation of CSCC tumorigenesis. On the whole, miR-3619-5p inhibited cell proliferation and cisplatin resistance of CSCC by regulating KPNA4 expression, suggesting that miR-3619-5p/KPNA4 pathway may represent a potential promising strategy for the treatment of patients with CSCC.

Microfluidic device for the analysis of MDR cancerous cell-derived exosomes' response to nanotherapy.

Exosomes are membrane-enclosed extracellular vesicles which have been indicated as important biomarkers of cancerous cell functionality, such as multiple drug resistance (MDR). Nanoparticles based chemotherapy is a promising strategy to overcome MDR by interfering the production and composition of exosomes. Therefore, tumor-derived exosomes post-treatment by nanotherapy are implied to play critical roles of biomarkers on cancer MDR analysis. However, the efficient isolation of such exosomes from extracellular environment for their therapeutic response analysis remains challenging. In this study, we presented a microfluidic device featured exosome specific anti-CD63 immobilized ciliated micropillars, which were capable to isolate cancer-derived exosomes from cell culture medium. The captured exosomes can be recovered intact by dissolving the cilia on the micropillars using PBS soaking. Owing to the immobilized antibody in the microfluidic device, nearly 70% of exosome from the biofluid could be isolated. So the secreted exosomes of the MDR and ordinary human breast cancer cells pre-treated by free drug or nanotherapy could be isolated with high purity. The drug contents of the isolated exosomes were measured to analysis of the exosomal pathway response of MDR cells to different chemotherapeutic formulations. Such analyses and further definition of the biomarkers of these exosomes could benefit the future investigations of accurately and reliably determine design principle, functional activity, and mechanisms of nanotherapy for MDR overcoming.

Multifunctional approaches utilizing polymeric micelles to circumvent multidrug resistant tumors.

The concerns impeding the success of chemotherapy in cancer is descending efficacy of drugs due to the development of multiple drug resistance (MDR). The current efforts employed to overcome MDR have failed or are limited to only preliminary in-vitro investigations. Nanotechnology is at the forefront of the drug delivery research, playing pivotal role in chemotherapy and diagnosis, thereby providing innovative approaches for the management of the disease with minimal side effects. Recently, polymeric micelles (PMs) have witnessed significant developments in cancer therapy. PMs are self-assembled colloidal particles, with a hydrophilic head and a long hydrophobic tail, which enhance the solubility, permeability and bioavailability of drugs, due to the unique features of reaching higher concentration in the biological system, above critical micellar concentration. One of the effective approaches to improve the efficacy of chemotherapy and overcome drug resistance would be to employ multifunctional approach (combination of stimuli-responsive, utilization of drug resistance modulators and combination therapy) using PMs as drug delivery systems. Actively targeted, stimuli-sensitive and multifunctional approaches involve using single and/or combination of approaches (pH-responsive, temperature regulated, reduction-sensitive, ultrasound etc.) to combat drug resistant. The review will describe PMs, types of copolymers used in PMs, preparation and characterization of PMs. A comprehensive list of PMs tested in clinical trials is discussed. Lastly, this review covers stimuli-sensitive and multifunctional approaches to overcome MDR in cancer utilizing PMs.

A polypeptide based podophyllotoxin conjugate for the treatment of multi drug resistant breast cancer with enhanced efficiency and minimal toxicity

Podophyllotoxin (PPT) is a chemotherapeutic agent which has shown significant activity against P-glycoprotein (P-gp) mediated multi drug resistant cancer cells. However, because of the poor aqueous solubility and high toxicity, PPT cannot be used in clinical cancer therapy. In order to enhance the efficiency and reduce side effect of PPT, a polypeptide based PPT conjugate PLG-g-mPEG-PPT was developed and used for the treatment of multi drug resistant breast cancer. The PLG-g-mPEG-PPT was prepared by conjugating PPT to poly(l-glutamic acid)-g-methoxy poly(ethylene glycol) (PLG-g-mPEG) via ester bonds. The PPT conjugates self-assembled into nanoparticles with average sizes about 100 nm in aqueous solution. Western blotting assay showed that the PLG-g-mPEG-PPT could effectively inhibit the expression of P-gp in the multiple drug resistant MCF-7/ADR cells. In vitro cytotoxicity assay indicated that the resistance index (RI) values of PLG-g-mPEG-PPT on different drug-resistant cancer cell lines exhibited 57–270 folds reduction than of traditional microtubule inhibitor chemotherapeutic drug PTX or DTX. Hemolysis assay demonstrated that the conjugation greatly decreased the hemolytic activity of free PPT. Maximum tolerated dose (MTD) of PLG-g-mPEG-PPT increased greatly (13.3 folds) as compared to that of free PPT. In vivo study showed that the PLG-g-mPEG-PPT conjugate remarkably enhanced the antitumor efficacy against MCF-7/ADR xenograft tumors with a tumor suppression rate (TSR) of 82.5%, displayed significantly improved anticancer efficacy as compared to free PPT (TSR = 37.1%) with minimal toxicity when both of the two formulations were used in MTD. Statement of SignificanceThe development of multiple drug resistance (MDR) of cancer cells is the main cause of chemotherapy failure. The over-expression of P-glycoprotein (P-gp) has been recognized to be the most important cause of MDR in cancer. Podophyllotoxin (PPT) is a chemotherapeutic agent which has shown strong activity against P-gp mediated multidrug resistant cancer cells by simultaneously inhibiting the over-expression of P-gp and the growth of cancer cells. However, PPT can not be used in clinical cancer treatment due to its poor aqueous solubility and high toxicity. Herein, we developed a polypeptide based PPT conjugate PLG-g-mPEG-PPT by conjugating PPT to poly(l-glutamic acid)-g-methoxy poly(ethylene glycol). The PLG-g-mPEG-PPT shows significantly decreased hemolytic activity, greatly improved maximum tolerated dose and remarkably enhanced antitumor efficacy against MCF-7/ADR xenograft tumors as compared to free PPT.

Aminoglucose-functionalized, redox-responsive polymer nanomicelles for overcoming chemoresistance in lung cancer cells

BackgroundChemotherapeutic drugs used for cancer therapy frequently encounter multiple-drug resistance (MDR). Nanoscale carriers that can target tumors to accumulate and release drugs intracellularly have the greatest potential for overcoming MDR. Glucose transporter-1 (GLUT-1) and glutathione (GSH) overexpression in cancer cells was exploited to assemble aminoglucose (AG)-conjugated, redox-responsive nanomicelles from a single disulfide bond-bridged block polymer of polyethylene glycol and polylactic acid (AG-PEG-SS-PLA). However, whether this dual functional vector can overcome MDR in lung cancer is unknown.ResultsIn this experiment, AG-PEG-SS-PLA was synthetized successfully, and paclitaxel (PTX)-loaded AG-PEG-SS-PLA (AG-PEG-SS-PLA/PTX) nanomicelles exhibited excellent physical properties. These nanomicelles show enhanced tumor targeting as well as drug accumulation and retention in MDR cancer cells. Caveolin-dependent endocytosis is mainly responsible for nanomicelle internalization. After internalization, the disulfide bond of AG-PEG-SS-PLA is cleaved in the presence of high intracellular glutathione levels, causing the hydrophobic core to become a polar aqueous solution, which subsequently results in nanomicelle disassembly and the rapid release of encapsulated PTX. Reduced drug resistance was observed in cancer cells in vitro. The caspase-9 and caspase-3 cascade was activated by the AG-PEG-SS-PLA/PTX nanomicelles through upregulation of the pro-apoptotic proteins Bax and Bid and suppression of the anti-apoptotic protein Bcl-2, thereby increasing apoptosis. Furthermore, significantly enhanced tumor growth inhibition was observed in nude mice bearing A549/ADR xenograft tumors after the administration of AG-PEG-SS-PLA/PTX nanomicelles via tail injection.ConclusionsThese promising results indicate that AG-PEG-SS-PLA/PTX nanomicelles could provide the foundation for a paradigm shift in MDR cancer therapy.

D-α-Tocopheryl Succinate/Phosphatidyl Ethanolamine Conjugated Amphiphilic Polymer-Based Nanomicellar System for the Efficient Delivery of Curcumin and To Overcome Multiple Drug Resistance in Cancer.

Nanomedicines have emerged as a promising treatment strategy for cancer. Multiple drug resistance due to overexpression of various drug efflux transporters and upregulation of apoptotic inhibitory pathways in cancer cells are major barriers that limit the success of chemotherapy. Here, we developed a d-α-tocopherol (α-TOS)/lipid-based copolymeric nanomicellar system (VPM) by conjugating phosphatidyl ethanolamine (PE) and α-TOS with poly(ethylene glycol) (PEG) via an amino acid linkage. The synthesized polymers were characterized by Fourier transform IR, gas-phase chromatography, and 1H and 13C NMR spectroscopy. VPM exhibited mean hydrodynamic diameter of 141.0 ± 0.94 nm with low critical micelles concentrations (CMC) of 15 μM compared to plain PEG-PE micelles (PPM) with size of 23.9 ± 0.34 nm and CMC 20 μM. The bigger hydrophobic compartment in VPM resulted in improved loading of a potent chemotherapeutic drug, curcumin (Cur), and increased encapsulation efficiency (EE) (% drug loading 98.3 ± 1.92, and 85.3 ± 3.29; EE 14.8 ± 0.16 and 12.8 ± 0.09 for VPM and PPM, respectively). Curcumin loaded Vitamin E based micelles exhibited higher cytotoxicity compared to Curcumin loaded PEG-PE micelles in tested cancer cell lines. C-VPM demonstrated ∼3.2 and ∼2.7-fold higher ability to reverse multiple drug resistance compared to PPM and verapamil (concentration used 30 μM), respectively. In the in vivo study by using B16F10 implanted C57Bl6/J mice, C-VPM reduced the tumor volume and weight more efficiently than C-PPM by inducing apoptosis as analyzed by TUNEL assay on tumor cryosections. The newly developed polymeric micelles, VPM with improved drug loadability and ability to reverse the drug resistance could successfully be utilized as a nanocarrier system for hydrophobic chemotherapeutic agents for the treatment of drug-resistant solid tumors.