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Purpose Given that prognosis of hepatocellular carcinoma (HCC) differs dramatically, it is imperative to uncover effective and available prognostic biomarker(s). The intratumor microbiome plays a significant role in the response to tumor microenvironment, we aimed to identify an intratumor microbiome signature for predicting the prognosis of HCC patients accurately and investigate its possible mechanisms subsequently.Methods The TCGA HCC microbiome data (TCGA-LIHC-microbiome) was downloaded from cBioPortal. To create an intratumor microbiome related prognostic signature, univariate and multivariate Cox regression analyses were used to quantify the association of microbial abundance and patients’ overall survival (OS), as well as their diseases specific survival (DSS). The performance of the scoring model was evaluated by the area under the ROC curve (AUC). Based on the microbiome related signature, clinical factors, and multi-omics molecular subtypes on the basis of “icluster” algorithm, nomograms were established to predict OS and DSS. Patients were further clustered into three subtypes based on their microbiome related characteristics by consensus clustering. Moreover, deconvolution algorithm, weighted correlation network analysis (WGCNA) and gene set variation analysis (GSVA) were used to investigate the potential mechanisms.Results In TCGA LIHC microbiome data, the abundances of 166 genera among the total 1406 genera were considerably associated with HCC patients’ OS. From that filtered dataset we identified a 27-microbe prognostic signature and developed a microbiome related score (MRS) model. Compared with those in relatively low risk group, patients in higher risk group own a much worse OS(P Conclusion MRS, a 27 intratumor microbiome related prognostic model, was successfully established to predict HCC patients overall survive independently. And the possible underlying mechanisms were also investigated to provide a potential intervention strategy.

Supplementary Figure 1, Table 1 from Natural product derivative Bis(4-fluorobenzyl)trisulfide inhibits tumor growth by modification of β-tubulin at Cys 12 and suppression of microtubule dynamics

Bis(4-fluorobenzyl)trisulfide (BFBTS) is a synthetic molecule derived from a bioactive natural product, dibenzyltrisulfide, found in a subtropical shrub, Petiveria allieacea. BFBTS has potent anticancer activities to a broad spectrum of tumor cell lines with IC50 values from high nanomolar to low micromolar and showed equal anticancer potency between tumor cell lines overexpressing multidrug-resistant gene, MDR1 (MCF7/adr line and KBv200 line), and their parental MCF7 line and KB lines. BFBTS inhibited microtubule polymerization dynamics in MCF7 cells, at a low nanomolar concentration of 54 nmol/L, while disrupting microtubule filaments in cells at low micromolar concentration of 1 μmol/L. Tumor cells treated with BFBTS were arrested at G2-M phase, conceivably resulting from BFBTS-mediated antimicrotubule activities. Mass spectrometry studies revealed that BFBTS bound and modified β-tubulin at residue Cys12, forming β-tubulin-SS-fluorobenzyl. The binding site differs from known antimicrotubule agents, suggesting that BFBTS functions as a novel antimicrotubule agent. BFBTS at a dose of 25 mg/kg inhibited tumor growth with relative tumor growth rates of 19.91%, 18.5%, and 23.42% in A549 lung cancer, Bcap-37 breast cancer, and SKOV3 ovarian cancer xenografts, respectively. Notably, BFBTS was more potent against MDR1-overexpressing MCF7/adr breast cancer xenografts with a relative tumor growth rate of 12.3% than paclitaxel with a rate of 43.0%. BFBTS displays a novel antimicrotubule agent with potentials for cancer therapeutics. [Mol Cancer Ther 2009;8(12):3318–30]

Polyethylene glycol (PEG)-conjugated lipid significantly contributed to the success of three approved lipid nanoparticles (LNP)-delivered therapeutics, including two COVID-19 mRNA vaccines. With the large-scale vaccination of mRNA vaccines, it has become an imminent task to elucidate the possible PEG-associated immune responses induced by clinically relevant LNP. Up to date there are only four small-scale population-based studies emphasizing the changes of PEG-specific antibodies upon injection of mRNA vaccines. However, inconsistent data were obtained due to significant person-to-person and study-to-study variabilities. To clarify the PEG-associated immune responses triggered by clinically relevant LNP in a model system with least “noise”, we initiated an animal study using the PEGylated LNP of BNT162b2 (with the largest number of recipients) as a representative LNP and simulated the clinical practice. Through designing a series of time points and three doses correlated with the PEG exposure amount contained in three approved LNP-based drugs, we demonstrated for the first time that generation and changes of anti-PEG IgM and IgG were time- and dose-dependent. Unexpectedly, we found that unlike other thymus-independent antigens (TI-Ag), PEGylated LNP not only induced isotype switch and production of anti-PEG IgG, but caused immune memory, leading to rapid enhancement and longer lasting time of both anti-PEG IgM and IgG upon repeated injection. Importantly, pharmacokinetic studies discovered that initial injection of PEGylated LNP accelerated the blood clearance of subsequently injected LNP. These findings refine our understandings on PEGylated LNP and possibly other PEG derivatives, and may lead to optimization of premarket guidelines and clinical practice of PEGylated LNP-delivered therapeutics.

Colorectal cancer (CRC) is the second most common cancer in men and the third most common cancer in women. Although long-term survival has improved over the past 30 years, at least 50% of patients with CRC will develop metastases after diagnosis. In this study, we examined whether quantifying the mRNA of six CRC-related genes in the blood could improve disease assessment through detection of circulating tumor cells (CTC), and thereby improve progression prediction in relapsed CRC patients. Cell spiking assay and RT-PCR were performed with blood samples from healthy volunteers spiked with six CRC cell lines to generate an algorithm, herein called the Six-gene Assay, based on six genes (CEA, EpCAM, CK19, MUC1, EGFR and C-Met) for CTC detection. The CTCs of 50 relapsed CRC patients were then respectively measured by CEA Gene Assay (single-gene assay control) and Six-gene Assay. Subsequently, receiver operating characteristic analysis of the CTC panel performance in diagnosing CRC was conducted for both assays. Moreover, the 2-year progression-free survival (PFS) of all patients was collected, and the application of CEA Gene Assay and Six-gene Assay in predicting PFS was carefully evaluated with different CTC cutoff values. Encouragingly, we successfully constructed the first multiple gene-based algorithm, named the Six-gene Assay, for CTC detection in CRC patients. Six-gene Assay was more sensitive than CEA Gene Assay; for instance, in 50 CRC patients, the positive rate of Six-gene Assay in CTC detection was 82%, whereas that of CEA Gene Assay was only 70%. Moreover, Six-gene Assay was more sensitive and accurate than CEA Gene Assay in diagnosing CRC as well as predicting the 2-year PFS of CRC patients. Statistical analysis demonstrated that CTC numbers measured by Six-gene Assay were significantly associated with 2-year PFS. This novel Six-gene Assay improves the definition of disease status and correlates with PFS in relapsed CRC, and thus holds promise for future clinical applications.

Clinical development of 7-ethyl-10‑hydroxy-camptothecin (SN38), the active metabolite of irinotecan (CPT-11), is hindered by its insolubility and poor stability. Another obstacle is that tumors could become resistant to SN38/CPT-11 through multiple mechanisms involving breast cancer resistance protein (BCRP). Herein one of the most potent and selective BCRP inhibitors, Ko143, is encapsulated into a recently constructed prodrug PEG-S-S-SN38 displaying a high and fixed drug loading, multiple intratumoral stimuli (oxidative stress, GSH and esterase)-responsive drug release and significant in vitro and in vivo superiorities over CPT-11. The obtained “combo” for simultaneous delivery of SN38 and Ko143, named as BI@PEG-SN38, has a high SN38 loading efficacy (14.85 wt.%) and a good Ko143 encapsulation efficacy (3.79%). Through generating panels of human colorectal cancer models expressing altered levels of BCRP via lentiviral transfection and CRISPR-Cas9, characteristics of different drug formulations are carefully evaluated. Impressively, BI@PEG-SN38 nanoparticles effectively reverse chemoresistance to CPT-11 (resistance index dropping from ∼274.00–456.00 to ∼1.70–4.68) and PEG-S-S-SN38 (resistance index dropping from ∼5.83–14.00 to ∼1.70–4.68) in three BCRP-overexpressing cancer cell lines. More importantly, reversal of BCRP-mediated chemoresistance to CPT-11 (P values lower than 0.001–0.0001) and PEG-S-S-SN38 (P values lower than 0.01–0.001) by BI@PEG-SN38 nanoparticles are further confirmed with two panels of colorectal cancer xenograft models in vivo. As the first nano-formulation of Ko143 and the first systemic co-delivery vehicle of SN38/CPT-11 and a BCRP inhibitor, BI@PEG-SN38 provides a new approach for resolving the bottlenecks for clinical translation of SN38 and numerous “chemosensitizers” like Ko143, and exhibits promising applicability in precision cancer medicine. Statement of significance To resolve the bottlenecks in clinical application of anticancer agents SN38/CPT-11 and the most potent breast cancer resistant protein (BCRP) inhibitor Ko143, a “combo” nanotherapeutic simultaneously delivering SN38 and Ko143 was constructed and named as BI@PEG-SN38. By generating panels of colorectal cancer models, we demonstrate that BI@PEG-SN38 nanoparticles effectively and selectively reversed BCRP-mediated tumor resistance to SN38/CPT-11 in vitro and in vivo. As the first nano-formulation of Ko143 and the first systemic co-delivery vehicle of SN38/CPT-11 and a BCRP inhibitor, BI@PEG-SN38 provides a new strategy for clinical development of SN38 and numerous “chemosensitizers”, and exhibits promising applicability in precision cancer medicine. Panels of cancer cell lines established here provides a useful platform for BCRP- and cancer-related research and technology development.

Accumulative data demonstrate that during the initiation and progression of tumors, several types of cellular components in tumor microenvironment, including tumor cells and immune cells, exhibit malfunctions in cellular energy metabolism. For instance, lipid metabolism impairments in immune cells are crucial in coordinating immunosuppression and tumor immune escape. In particular, excessive lipids have been shown to exhibit negative effects on innate immunity. Previous studies on lipid metabolism in immune cells are mainly focused on macrophages and T lymphocytes. Although natural killer (NK) cells are major players in the innate elimination of virus, bacteria, and tumor cells, available literature reports related with lipid metabolism in NK cells and tumor-associated NK (TANK) cells are very sparse. Despite these, the importance and clinical relevance of the crosstalk among lipid metabolism, NK/TANK cells, and tumors have been clearly indicated. In this chapter, following a general description of NK and TANK cells, our knowledge on the regulation of lipid metabolism in NK cells is reviewed, with an emphasis on the roles of mTOR and SREBP signaling. Then the interactions between lipid metabolism and NK/TANK cells under pathological conditions, e.g., obesity and cancer, were carefully introduced. As there is an urgent need to reveal more regulators and to clarify detailed molecular mechanisms by which lipid metabolism interacts with NK/TANK cells, several categories of potential regulators/pathways, as well as the challenges and perspectives in this emerging field, are discussed.

Background: Parturition involves the transformation of the quiescent myometrium to the laboring state, which is driven by changes in myometrial gene expression. This study aimed to identify myometrial transcriptomic signatures and potential novel hub genes in parturition, which have great significance for understanding successful parturition and treating labor pathologies such as preterm birth. Methods: In our study, human myometrium was collected from women in spontaneous term labor (TL=8) and term not in labor (TNL=8) to survey transcriptional changes associated with labor by using RNA-seq. We screened the differentially expressed genes (DEGs) of functional enrichment analysis including GO and KEGG pathway analysis and gene set enrichment analysis (GSEA) to study the role of DEGs and potential underlying mechanisms of parturition. Moreover, we used protein-protein interactions network analysis to recognize the hub genes among these DEGs. Furthermore, the biological behavior of hub genes in myometrial contraction was verified by cellular experiments. Findings: A total of 582 DEGs were selected. The enrichment analysis revealed that DEGs were enriched in signal transduction, regulation of signaling receptor activity, inflammatory response, cytokine-cytokine receptor interaction, IL-17 signaling pathway, TNF signaling pathway and so on. FPR1 was highly expressed in laboring myometrium. The experimental results showed that activating FPR1 in human myometrial cells, resulted in increased myometrial contraction. Interpretation: The transcriptome analysis of term labor myometrium revealed that labor onset was associated with inflammatory response. Our findings implicate the novel role of FPR1 as a modulator of myometrial contraction. Funding: This study was supported by the National Key Research and Development Program of China (No.2018YFC1004603). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: This study was approved by the Ethics Committee of Women’s Hospital, School of Medicine, Zhejiang University

Accumulative data demonstrate that during the initiation and progression of tumors, several types of cellular components in tumor microenvironment, including tumor cells and immune cells, exhibit malfunctions in cellular energy metabolism. For instance, lipid metabolism impairments in immune cells are crucial in coordinating immunosuppression and tumor immune escape. In particular, excessive lipids have been shown to exhibit negative effects on innate immunity. Previous studies on lipid metabolism in immune cells are mainly focused on macrophages and T lymphocytes. Although natural killer (NK) cells are major players in the innate elimination of virus, bacteria, and tumor cells, available literature reports related with lipid metabolism in NK cells and tumor-associated NK (TANK) cells are very sparse. Despite these, the importance and clinical relevance of the crosstalk among lipid metabolism, NK/TANK cells, and tumors have been clearly indicated. In this chapter, following a general description of NK and TANK cells, our knowledge on the regulation of lipid metabolism in NK cells is reviewed, with an emphasis on the roles of mTOR and SREBP signaling. Then the interactions between lipid metabolism and NK/TANK cells under pathological conditions, e.g., obesity and cancer, were carefully introduced. As there is an urgent need to reveal more regulators and to clarify detailed molecular mechanisms by which lipid metabolism interacts with NK/TANK cells, several categories of potential regulators/pathways, as well as the challenges and perspectives in this emerging field, are discussed.

The clinical utility of doxorubicin (DOX) is restricted by its severe side effects. Continuous efforts are aimed at developing efficacious DOX-delivery systems that may overcome the drawbacks of existing ones. Herein, we report a self-assembling prodrug forming high drug loading nanoparticles for DOX delivery. A low molecular weight polyethylene glycol (PEG) chain as the hydrophilic part was anchored to hydrophobic DOX via an acid-cleavable hydrazone bond to form the amphiphilic prodrug PEG–DOX. In aqueous solution, PEG–DOX formed nanoparticles with a diameter of ∼125 nm and extremely high drug loading (∼46 wt%). These nanoparticles were stable in PBS but released DOX in an acidic pH-triggered manner. Interestingly, taken up by cells via endocytosis, PEG–DOX bypassed the P-glycoprotein (P-gp)-mediated efflux of DOX, leading to drug accumulation in DOX-resistant human breast cancer cells (MCF-7/ADR). More importantly, PEG–DOX exhibited potent antitumor activity in vitro and in vivo, and showed significantly increased in vivo safety than free DOX. These encouraging data merit further preclinical and clinical development on PEG–DOX.

Modification of drug delivery systems (DDSs) with stimuli-responsive elements could significantly increase the tumor-specific delivery of anticancer drugs. Herein we synthesized a novel multiple stimuli-responsive SN38 prodrug, named PEG-S-S-SN38, by conjugating PEG (MW: 2000) and SN38 with disulfide bonds and carbonic ester linkages as linkers for efficient delivery of SN38. The amphiphilic PEG-S-S-SN38, with a high SN38 loading content, could self-assemble into nanoparticles (NPs) with a stable diameter of ∼73 nm. PEG-S-S-SN38 NPs release SN38 very slowly at physiological pH, while they quickly release SN38 in the presence of GSH, esterase and H2O2, all of which are abundant in the cytoplasm of cancer cells. PEG-S-S-SN38 NPs could be quickly internalized into tumor cells, achieve vesicular escape and nuclear localization, and exhibit remarkable in vitro anticancer activity similar to SN38. Encouragingly, PEG-S-S-SN38 NPs exhibit the same effects on cell cycle regulations as SN38 in vitro. Most importantly, the inhibition rate of tumor growth induced by PEG-S-S-SN38 NPs in a xenograft tumor model reached 72.49% ± 6.26%, which was nearly double that of the corresponding clinical drug CPT-11 (38.64% ± 13.04%) at a dosage equivalent to 10 mg kg−1 SN38. Our data suggest that the multi-stimuli responsiveness of PEG-S-S-SN38 NPs remarkably enhances their therapeutic activity against heterogeneous or mixed cell population in tumors, making this new DDS a promising alternative to CPT-11 for cancer treatment.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a dismal overall prognosis mainly unchanged over the past decades. PDAC is generally refractory to conventional treatments, and thus novel therapies are urgently needed. Recently, accumulating evidence has indicated that human pancreatic stellate cells (PSCs) facilitate PDAC development and drug resistance through paracrine activation of hedgehog pathway. Here, we report that smart SN38 (active metabolite of irinotecan) polymeric prodrug-based nanoparticles effectively encapsulate the commercial hedgehog pathway inhibitor GDC-0449 for co-delivery. More intriguingly, we obtained size-tunable nanoparticles with increased GDC-0449 loading efficiency by simply extending the chain length of the hydrophobic SN38 block. To better evaluate the efficacy and investigate the synergistic mechanisms, we immortalized human PSCs and established fibroblast-containing models in vitro and in vivo. In PSCs, BxPC-3 cells and MIA PaCa-2 cells, GDC-0449 suppressed the co-culture induced up-regulations of the two drug resistance contributors: sonic hedgehog transcription factor glioma-associated protein1 (GLI-1) and UGT1A glucuronosyltransferase. Importantly, the nanoparticle-mediated co-delivery system exhibited potent antitumor efficacy with enhanced apoptosis and reduced collagen, α-SMA and GLI-1 expression in tumor tissues. These findings reveal a potential strategy to utilize nanoparticle-mediated drug co-delivery platform as an effective combination therapy for fibroblast-enriched PDAC.

Multi-arm star poly(ethylene glycol) (PEG) polymers have a number of advantages over linear PEG polymers when used as carriers for drug delivery and controlled release. For instance, they have more terminals that can be modified to form multi-functional delivery systems with significantly increased drug loading. They can form micelles with higher stability and lower critical micelle concentration (CMC), which helps to improve the blood circulation and reduce the unfavorable burst drug release. Moreover, star PEG polymers can form three-dimensional hydrogels with controllable size and adjustable functions through cross-linking. Indeed, these unique advantages of star PEG polymers have promoted investigations on star PEG-based drug delivery systems. Herein, for the first time, we carefully reviewed the advances on the research and development of star PEG polymers, especially the 4-, 6- and 8-armed star PEG polymers, in delivery and controlled release of a series of bioactive agents, including both small molecules and biomacromolecules. Opportunities and challenges for successful translation of star PEG-based drug formulations into clinical use were also discussed.

The significant disparities in metabolism between tumor and normal cells have inspired the development of metabolism-based anti-tumor therapeutics. Arginine is a semi-essential amino acid because normal cells can not only synthesize arginine de novo but also take up extracellular arginine. Several types of tumors have abnormalities in arginine metabolism enzymes and completely rely on extracellular arginine to support necessary biological processes. This property is referred to as arginine auxotrophy. Taking advantage of characteristic arginine auxotrophy in tumors, arginine deprivation, which is generally induced by the use of arginine deiminase (ADI) and arginase I, has been investigated as a novel strategy for cancer therapy. Arginine deprivation demonstrated promising efficacy against arginine-auxotrophic tumors. By integrating perspectives from both clinical oncologists and laboratory scientists, this article reviews the important aspects of arginine deprivation as a promising anticancer therapy.

Guanidine was introduced to low molecular weight linear polyethyleneimine (LPEI) via amide groups, to explore the effect of both guanidine degree and pendant chain length on its transfection behavior. The resulting guanidinoamidized LPEIs (GLPEIs) could dramatically reduce LPEI’s toxicity, enhance its DNA-packaging capability, cellular uptake and therefore transfection efficiency. These polyplexes were taken up very efficiently via caveolae-mediated endocytosis and their transfection efficiencies in ovarian cancer cells were significantly improved compared to native LPEI10k polyplexes. Among these GLPEIs, LPEI-C3-G100 showed higher DNA affinity even than LPEI25k and the highest transfection efficiency, probably due to the optimization of polymer chain flexibility. Of notice, LPEI-C3-G100 polyplexes could more effectively accumulate into cytoplasm than LPEI25k, although the transfection efficiency of LPEI-C3-G100 polyplexes was not superior to that of LPEI25k polyplexes, which would be probably attributed to the more efficient release of LPEI25k polyplexes than LPEI-C3-G100 polyplexes in the cytoplasm.

The use of binding materials in ancient Chinese textiles was widely practiced to enhance both the bond strength and the cohesion between fibers. An accurate qualitative assessment of such binding materials is of great significance in heritage research and conservation science: first, to understand the historical development of the textile industry and, second, to choose a suitable conservation method. However, this problem has not been fully addressed due to the complexity of the binder composition and the limitations of traditional analytical methods. This study focused on the use of enzyme-linked immunosorbent assay (ELISA) for the specific identification of proteins in archaeological binder materials. Four ancient Chinese textile binder samples were tested by ELISA. The results showed that all the binder samples reacted positive for ovalbumin, while two printing gold samples reacted negative for collagen-I. This is the first time ELISA has been successfully used to detect protein in ancient Chi...

The stability and size of polymeric nanoparticles are two of the most important parameters determining their pharmacokinetics and tumor/drug accumulation efficiency in cancer-drug delivery. Herein, we report a facile one-pot synthesis of crosslinked nanoparticles (CNPs) with tunable sizes and polyethylene glycol (PEG) shells via click reactions. Simply by adjusting the contents of the macromonomer (PEG monoacrylate) in its reaction with ethylene diacrylate and a crosslinker containing hexa-thiols groups, the sizes of the resulting PEGylated crosslinked nanoparticles could be easily tuned from 10 to 90 nm. These nanoparticle cores could encapsulate hydrophobic drugs such as doxorubicin (DOX), and the unreacted thiol and acrylate groups could be used for drug conjugation or labeling. Thus, the nanoparticles provide a multifunctional platform for drug delivery. In vivo studies showed that the larger nanoparticles (about 83.7 nm) had a much longer blood-circulation time and better tumor-targeting efficiency. One of our most important findings was that the drug encapsulated in the crosslinked nanoparticles, even though little was released at pH 7.4 under in vitro conditions, had much faster blood clearance than the nanoparticles’ carrier, suggesting that drug release in the bloodstream was significant.

Novel classes of jellyfish-shaped amphiphilic dendrimers composed of 7 hydrophilic poly(ethylene glycol) (PEG) arms and 14 hydrophobic polyester dendrons with β-cyclodextrin (βCD) as the core molecule were synthesized by a facile method. Seven PEG chains were first conjugated to the C-6 positions of native βCD. Subsequently, dendritic polyester architectures were constructed from the remaining 14 secondary hydroxyl groups at C-2 and C-3 positions of the βCD moiety, resulting in jellyfish-shaped amphiphilic dendrimers of different generations (7PEG-βCD-Gx) with well-defined molecular structures. The amphiphilic dendrimers self-assembled into different morphologies dependent upon the hydrophilic fraction of the dendrimers, and very surprisingly, the fourth-generation dendrimers consisting of only several percent of PEG could form aggregates with extremely narrow size distributions.

Highly sensitive and safe contrast agents (CAs) are essential for magnetic resonance imaging (MRI) to achieve accurate tumor detection and imaging. Dendrimer-based macromolecular MRI contrast agents are advantageous owing to their tumor-targeting ability, enhanced imaging contrast and enlarged imaging window. However, most of them have drawbacks of non-degradability and thereby long-term retention in body and toxicity. Herein, a tumor-targeting biodegradable dendritic CA (DCA) (FA-PEG-G2-DTPA-Gd) was prepared from a polyester dendrimer conjugated with gadolinium (Gd) chelates and PEG chains with distal folic acid. The DCA had a high longitudinal relaxivity up to 17.1 mM − 1 s − 1 , 4 times higher than the clinically used CA Magnevist. The MRI contrasted by FA-PEG-G2-DTPA-Gd outlined the inoculated tumor more clearly, and had much higher contrast enhancement for a much longer time than Magnevist. More importantly, the biodegradable FA-PEG-G2-DTPA-Gd gave much less Gd retentions in all the organs or tissues than non-degradable DCAs. Thus, the high efficiency in MRI contrast enhancement and low Gd retention merit it a promising CA for contrast enhanced tumor MRI.

A prodrug forms nanocapsules responsive to tumor GSH/ROS heterogeneity releasing the parent drug SN38 via thiolysis in the presence of GSH (glutathione) or via enhanced hydrolysis due to ROS (reactive oxygen species)-oxidation of the linker, giving rise to high in vitro cytotoxicity and in vivo anticancer therapeutic activity. The nanocapsules are a suitable size for tumor targeting by means of the EPR effect and have a fixed SN38 loading content of 35 wt%, ideal for translational nanomedicine.

Drug resistance, a major obstacle to successful cancer chemotherapy, frequently occurs in recurrent or metastatic breast cancer and results in poor clinical response. Fulvestrant is a new type of selective estrogen receptor (ER) downregulator and a promising endocrine therapy for breast cancer. In this study, we evaluated the combination treatment of fulvestrant and doxorubicin in ER-negative multidrug-resistant (MDR) breast cancer cell lines Bads‑200 and Bats‑72. Fulvestrant potentiated doxorubicin-induced cytotoxicity, apoptosis and G2/M arrest with upregulation of cyclin B1. It functioned as a substrate for P-glycoprotein (P-gp) without affecting its expression. Furthermore, fulvestrant not only restored the intracellular accumulation of doxorubicin but also relocalized it to the nuclei in Bats‑72 and Bads‑200 cells, which may be another potential mechanism of reversal of P-gp mediated doxorubicin resistance. These results indicated that the combination of fulvestrant and doxorubicin-based chemotherapy may be feasible and effective for patients with advanced breast cancer.

Doxorubicin (DOX) is a widely used chemotherapeutic drug to treat a range of cancers. However, its unfavorable effects, particularly the cardiotoxicity and the induction of multidrug resistance (MDR), significantly limit its clinical applications. Herein, a novel doxorubicin prodrug, PEG2K -DOX, is synthesized by conjugating a deprotonated doxorubicin molecule with the polyethylene glycol (PEG, MW: 2K) chain via pH-responsive hydrazone bond, and its potential as a better alternative than doxorubicin is evaluated. The data show that the amphiphilic PEG2K -DOX can self-assemble into stable nanoparticles with a high and fixed doxorubicin loading content (≈20 wt%), a favorable size of 91.5 nm with a narrow polydispersity (PDI = 0.14), good stability, and pH-dependent release behavior due to the acid-cleavable linkage between PEG and doxorubicin. Although doxorubicin hardly accumulates in MDR cells, PEG2K -DOX nanoparticles significantly increase the cellular uptake and cell-killing activity of doxorubicin in two MDR cancer cell lines MCF-7/ADR and KBv200, with the IC50 values dropped to 1.130% and 42.467% of doxorubicin, respectively. More impressively, PEG2K -DOX nanoparticles exhibit significantly improved plasma pharmacokinetics, increased in vivo therapeutic efficacy against MDR xenograft tumors, and better in vivo safety compared with doxorubicin. PEG2K -DOX nanoparticles hold the promise to become a better alternative than doxorubicin for cancer treatment, especially for MDR tumors.

Platinum (IV)-coordinate polymers were synthesized by condensation polymerization using diamminedichlorodihydroxyplatinum (DHP) or its dicarboxyl derivative diamminedichlorodisuccinatoplatinum (DSP) as comonomers. Cyclic voltammogram study showed that Pt (IV) in the polymers was much easier reduced to Pt (II), particularly at the acidic pH, than that in the monomer DSP. Thus, these polymers were intracellular reduction-responsive backbone-type polymer conjugates that could be degraded and release Pt (II). These conjugates not only had high and fixed platinum contents (27.7% for P(DSP-EDA) and 29.6% for P(DSP-PA), respectively), but also showed increased cytotoxicity compared with corresponding Pt (IV) monomer DSP toward various tumor cell lines. In vivo, the conjugate showed a longer blood circulation time and better tumor accumulation.

Nanosystems with unique physical and biological properties have been extensively explored for cancer targeted intracellular delivery of small-molecular chemotherapeutic drugs to increase their therapeutic efficacies and to minimize their side effects. A large number of anticancer drugs are DNA-toxins that bind nuclear DNA or its associated enzymes to exert their cytotoxicity to cancer cells. After entering tumor cells, they need to be further delivered to the nucleus for actions. Herein, we discuss the biological barriers and summarize recent progress of nuclear drug delivery for cancer chemotherapy, emphasizing strategies that appear useful for design of vehicles capable of delivering drugs to the nucleus, particularly for in vivo applications. The existing obstacles or problems that need to be overcome before successful applications of nuclear drug delivery for cancer chemotherapy are also discussed.

A biodegradable tumor targeting nano-probe based on poly(ɛ-caprolactone)-b-poly(ethylene glycol) block copolymer (PCL-b-PEG) micelle functionalized with a magnetic resonance imaging (MRI) contrast agent diethylenetriaminepentaacetic acid-gadolinium (DTPA-Gd3+) on the shell and a near-infrared (NIR) dye in the core for magnetic resonance and optical dual-modality imaging was prepared. The longitudinal relaxivity (r1) of the PCL-b-PEGDTPA-Gd3+ micelle was 13.4 (mmol/L)−1s−1, three folds of that of DTPA-Gd3+, and higher than that of many polymeric contrast agents with similar structures. The in vivo optical imaging of a nude mouse bearing xenografted breast tumor showed that the dual-modality micelle preferentially accumulated in the tumor via the folic acid-mediated active targeting and the passive accumulation by the enhanced permeability and retention (EPR) effect. The results indicated that the dualmodality micelle is a promising nano-probe for cancer detection and diagnosis.

Poly(β-aminoester) dendrimers have been prepared. These systems represent the first degradable dual pH- and temperature-responsive dendrimers displaying photoluminescence. The pH/temperature sensitivities are interrelated; the lower critical solution temperature of the dendrimer decreases as the pH of the solution is increased. The sensitivities are mainly due to phase changes of the surface groups with changes in pH or temperature. These dual-responsive dendrimers are very useful in drug delivery. They may be loaded with a hydrophobic drug at low temperature without using organic solvents. The loaded drug is released very slowly and steadily at 37 °C and physiological pH, but can be quickly released at acidic pH, for example the lysosomal pH (pH 4-5), for intracellular drug release. These dendrimers also display strong photoluminescence, which can be exploited for monitoring drug loading and release. Thus, poly(β-aminoester) dendrimers constitute ideal drug carriers since their thermal sensitivity allows the loading of drugs without using organic solvents, their pH sensitivity permits fast intracellular drug release, and their photoluminescence provides a means of monitoring drug loading and release.

Gene therapy has emerged as a potential new approach to treat genetic disorders by delivering therapeutic genes to target diseased tissues. However, its clinical use has been impeded by gene delivery systems. The viral vectors are very efficient in delivering and expressing their carried genes, but they have safety issues in clinical use. While nonviral vectors are much safer with very low risks after careful material design, but their gene transcription efficiency is too low to be clinically used. Thus, rational design of nonviral vectors mimicking the viral vectors would be a way to break this bottleneck. This review compares side-by-side how viral/nonviral gene vectors transcend these biological barriers in terms of blood circulation, cellular uptake, endosome escape, nucleus import and gene transcription.

Aims: Polyamidoamine (PAMAM) dendrimers with primary amine termini have been extensively explored as drug and gene carriers owing to their unique properties, but their amine-carried cationic charges cause nonspecific cellular uptakes, systemic toxicity and other severe problems in in vivo applications. Method: In this article, we report a charge-reversal approach that latently deactivates PAMAM’s primary amines to negatively charged acid-labile amides in order to inhibit its nonspecific interaction with cells, but regenerates the active PAMAM once in acidic environments. Results: A cascade cancer cell nuclear drug delivery was achieved using the latently amidized PAMAM as the carrier conjugated with folic acid as the targeting group and a DNA-toxin drug camptothecin. The conjugate had low nonspecific interactions with cells, but easily entered cancer cells overexpressing folate receptors via receptor-mediated endocytosis. Subsequently, the endocytosed conjugate was transferred to acidic lysosomes, wherein the active PAMAM carrier was regenerated, escaped from the lysosome and then entered the nucleus for drug release. Conclusion: This reversible deactivation/activation makes PAMAM dendrimers useful nanocarriers for in vivo cancer cell nuclear-targeted drug delivery.

Bis(4-fluorobenzyl)trisulfide (BFBTS) is a synthetic molecule derived from a bioactive natural product, dibenzyltrisulfide, found in a subtropical shrub, Petiveria allieacea. BFBTS has potent anticancer activities to a broad spectrum of tumor cell lines with IC50 values from high nanomolar to low micromolar and showed equal anticancer potency between tumor cell lines overexpressing multidrug-resistant gene, MDR1 (MCF7/adr line and KBv200 line), and their parental MCF7 line and KB lines. BFBTS inhibited microtubule polymerization dynamics in MCF7 cells, at a low nanomolar concentration of 54 nmol/L, while disrupting microtubule filaments in cells at low micromolar concentration of 1 μmol/L. Tumor cells treated with BFBTS were arrested at G2-M phase, conceivably resulting from BFBTS-mediated antimicrotubule activities. Mass spectrometry studies revealed that BFBTS bound and modified β-tubulin at residue Cys12, forming β-tubulin-SS-fluorobenzyl. The binding site differs from known antimicrotubule agents, suggesting that BFBTS functions as a novel antimicrotubule agent. BFBTS at a dose of 25 mg/kg inhibited tumor growth with relative tumor growth rates of 19.91%, 18.5%, and 23.42% in A549 lung cancer, Bcap-37 breast cancer, and SKOV3 ovarian cancer xenografts, respectively. Notably, BFBTS was more potent against MDR1-overexpressing MCF7/adr breast cancer xenografts with a relative tumor growth rate of 12.3% than paclitaxel with a rate of 43.0%. BFBTS displays a novel antimicrotubule agent with potentials for cancer therapeutics. [Mol Cancer Ther 2009;8(12):3318–30]

The combination of carboplatin and paclitaxel is widely used to treat multiple solid tumors including ovarian, lung and breast cancer. Usually these drugs are given simultaneously with little regard to the importance of scheduling to obtain a maximal response. To investigate the importance of sequencing, the human breast Bcap37 and ovarian OV2008 cancer cell lines were exposed to carboplatin and paclitaxel in three different sequences: (1) pretreatment with paclitaxel followed by carboplatin, (2) pretreatment of carboplatin followed by paclitaxel and (3) simultaneous treatment with these two agents. The combination of carboplatin and paclitaxel resulted in antagonistic interactions when tumor cells were exposed to carboplatin prior to paclitaxel or exposed to the two drugs simultaneously, but there was little antagonistic interaction observed when paclitaxel was administered before carboplatin. Biochemical examination revealed that pretreatment or cotreatment of carboplatin inhibited paclitaxel-induced IkappaBalpha degradation and bcl-2 phosphorylation. Further analyses demonstrated that carboplatin could significantly interfere with the cytotoxic effects of paclitaxel on both mitotic arrest and apoptotic cell death unless paclitaxel was administered before carboplatin. These results indicate that the interaction between paclitaxel and carboplatin is highly schedule dependent. The optimal schedule for this combination is sequential exposure of paclitaxel followed by carboplatin.

The specific sizes that determine optimal nanoparticle tumor accumulation, penetration, and treatment remain inconclusive because many studies compared nanoparticles with multiple physicochemical variables (e.g., chemical structures, shapes, and other physical properties) in addition to the size. In this study, we synthesized amphiphilic block copolymers of 7-ethyl-10-hydroxylcamptothecin (SN38) prodrug and fabricated micelles with sizes ranging from 20 to 300 nm from a single copolymer. The as-prepared micelles had exactly the same chemical structures and similar physical properties except for size, which provided an ideal platform for a systematic investigation of the size effects in cancer drug delivery. We found that the micelle's blood circulation time and tumor accumulation increased with the increase in their diameters, with optimal diameter range of 100 to 160 nm. However, the much higher tumor accumulation of the large micelles (100 nm) did not result in significantly improved therapeutic efficacy, because the large micelles had poorer tumor penetration than the small ones (30 nm). An optimal size that balances drug accumulation and penetration in tumors is critical for improving the therapeutic efficacy of nanoparticulate drugs.

A key barrier to the development of gene therapy remains the lack of safe, efficient and easily controllable vehicles for gene delivery. The fundamental problems associated with the viral vehicles, e.g. lack of specificity and immunogenic potential, have driven the development of non-viral systems of gene delivery. In the last decade, studies on p53 gene replacement therapy have dominated the literature. Although clinical trials of p53 gene therapy have achieved limited success, it remains the only tumor suppressor gene to be evaluated formally in clinical trials for cancer treatment, with increasing focus on delivery using non-viral systems. In this article, we particularly review current investigations on p53 gene delivery using non-viral methods, including both physical and chemical approaches, with an emphasis on the latter. The existing opportunities and challenges for successful p53 cancer gene therapy are also discussed.

Paclitaxel is a widely used naturally occurring antineoplastic agent that has shown great promise in the treatment of a variety of human solid tumors, particularly for advanced breast and ovarian cancers. Recent studies in our laboratory discovered that glucocorticoids could selectively inhibit paclitaxel-induced apoptosis in a number of human solid tumor cell lines in vitro. Since glucocorticoids (such as dexamethasone) are routinely used as a premedication in the clinical application of paclitaxel to prevent hypersensitivity reactions and other adverse effects, the inhibitory effect of glucocorticoids on paclitaxel-induced apoptosis has raised a clinically relevant question as to whether the pretreatment with glucocorticoids might interfere with the therapeutic efficacy of paclitaxel. In the present study, through development of animal models bearing human breast and ovarian xenograft tumors, we evaluated the potential influence of dexamethasone on antitumor activity of paclitaxel in vivo. The results demonstrated that pretreatment of dexamethasone significantly attenuated therapeutic efficacy of paclitaxel against human breast and ovarian xenograft tumors. The inhibition rate of 20 mg paclitaxel/kg on the growth of breast and ovarian xenograft tumors was around 20-25% less when the animals were pretreated with 1 mg dexamethasone/kg. Further analyses with histological examination and TdT-mediated dUTP nick end labeling assay indicate that pretreatment with dexamethasone clearly interferes with the cytotoxic effects of paclitaxel on both morphological alterations and induction of cell death. Additionally, immunohistochemical staining of proliferation marker Ki-67 indicates that the percentage of proliferating cells in xenograft tumors with pretreatment of dexamethasone is much higher than that in the tumors treated with paclitaxel alone. Put together, the results obtained from the animal experiments show that pretreatment of xenograft tumors with dexamethasone results in significant inhibition of the therapeutic efficacy of paclitaxel in vivo. This finding may have a potential implication on the clinical practice of paclitaxel-based chemotherapy.

Purpose: The promising clinical activity of paclitaxel, a naturally occurring antimicrotubule agent, has promoted considerable interest in combining this drug with radiation therapy, but it remains unclear whether such a combination would increase the therapeutic efficacy. This study is to assess the potential interactions between paclitaxel and γ-radiation against human tumor cells in vitro. Experimental Design: Paclitaxel and γ-radiation were administered in three different sequences designated as pre-radiated, co-radiated, and post-radiated to BCap37 (human breast cancer cell line) and KB (human epidermoid carcinoma cell line) cells. The cytotoxic interactions between and mutual influences of these two agents on their antitumor activities were analyzed by a series of assays including cytotoxic, morphological, and biochemical examinations. Results: The combination of paclitaxel and γ-radiation did not produce a synergistic or additive effect. Instead, the overall in vitro cytotoxicity of these combinations was much lower than that of paclitaxel treatment alone. DNA fragmentation and flow cytometric assays showed that the addition of γ-radiation interfered with paclitaxel-induced apoptosis. Further analyses indicated that the addition of γ-radiation resulted in a transient or prolonged cell cycle arrest at G2 phase, which likely prevented the cytotoxic effects of paclitaxel on both mitotic arrest and apoptosis. In addition, biochemical examinations revealed that γ-radiation inhibited paclitaxel-induced IκBα degradation and bcl-2 phosphorylation and increased the protein levels of cyclin B1 and inhibitory phosphorylation of p34cdc2. Conclusions: Our results suggest that γ-radiation might specifically block the cell cycle at G2 phase, which in turn prevents the cytotoxic effects of paclitaxel on both mitotic arrest and apoptosis. Therefore, it eventually results in a cell cycle-dependent antagonistic effect on the antitumor activity of paclitaxel. This finding may be relevant to the clinical application of combination therapy with paclitaxel and radiation.

Antimicrotubule Vinca alkaloids, such as vinblastine and vincristine, interfere with the dynamics of microtubules and have shown significant cell killing activity in a variety of tumor cells through induction of apoptosis. The mechanism by which Vinca alkaloids induce apoptosis is not entirely clear. In this study, we found that glucocorticoids inhibit Vinca alkaloid-induced apoptosis without affecting G2-M arrest in human breast cancer BCap37 cells and human epidermoid tumor KB cells, suggesting that Vinca alkaloid-induced apoptosis may occur via a pathway independent of cell cycle arrest. Further analyses indicated that Vinca alkaloids cause significant degradation of IκBα, which in turn results in nuclear factor-κB (NF-κB) activation. Transfection of antisense IκBα in BCap37 cells sensitizes Vinca alkaloid-induced apoptosis. Moreover, in vitro kinase assays show that the activity of IκB kinase (IKK) was activated by Vinca alkaloids and was not affected by glucocorticoids. Stable transfection of dominant-negative deletional mutant IκBα, which is insensitive to IKK-mediated phosphorylation and degradation, resulted in the inhibition of Vinca alkaloid-induced NF-κB activation and reduced sensitivity of tumor cells to Vinca alkaloid-induced apoptosis. These findings suggest that the NF-κB/IκB signaling pathway may contribute to the mediation of Vinca alkaloid-induced apoptosis in human tumor cells.

The enhanced permeability and retention (EPR) effect of tumors is much more complex than initially defined, and it alone is not sufficient for targeted delivery of nanosized agents. Meanwhile, poor tumor penetration is another major challenge for the treatment of solid tumors using nanoparticles. Development of delivery systems for SN38, the active metabolite of CPT-11 in human and a very potent anticancer molecule, has become an attractive research area. PEGx -p(HEMASN38)y (x and y are viable), a prodrug synthesized by using polyethylene glycol (PEG) as initiator and SN38 as monomer through atom transfer radical polymeration (ATRP) method, is previously reported. Using PEG2.4K -p(HEMASN38)3K as a model prodrug, herein an active-targeted strategy decorated with cys-arg-gly-asp-lys (CRGDK), a peptide specifically binds to neuropilin-1 overexpressed by tumor vessels and tumor cells, is successfully established to further improve the delivery and efficacy of SN38. CRGDK-functionalized PEG2.4K -p(HEMASN38)3K (C-SN38) nanoparticles and nonfunctionalized control (B-SN38) are prepared with two distinct sizes, 30 and 100 nm. Their physiochemical and biological characteristics are investigated in vitro and in vivo with multiple tumor models. It is demonstrated for the first time that CRGDK functionalization can be a promising strategy for efficient delivery of SN38, and C-SN38 is a potent drug candidate for the treatment of neuropilin-1 overexpressing tumors.