Bifidobacterium longum SX-1326 ameliorates gastrointestinal toxicity after irinotecan chemotherapy via modulating the P53 signaling pathway and brain-gut axis.

BACKGROUND: Colorectal cancer (CRC) is a prevalent malignant malignancy affecting the gastrointestinal tract that is usually treated clinically with chemotherapeutic agents, whereas chemotherapeutic agents can cause severe gastrointestinal toxicity, which brings great pain to patients. Therefore, finding effective adjuvant agents for chemotherapy is crucial. METHODS: In this study, a CRC mouse model was successfully constructed using AOM/DSS, and the treatment was carried out by probiotic Bifidobacterium longum SX-1326 (B. longum SX-1326) in combination with irinotecan. Combining with various techniques of modern biomedical research, such as Hematoxylin and Eosin (H&E), Immunohistochemistry (IHC), Western blotting and 16S rDNA sequencing, we intend to elucidate the effect and mechanism of B. longum SX-1326 in improving the anticancer efficacy and reducing the side effects on the different levels of molecules, animals, and bacteria. RESULTS: Our results showed that B. longum SX-1326 enhanced the expression of Cleaved Caspase-3 (M vs. U = p < 0.01) and down-regulated the expression level of B-cell lymphoma-2 (Bcl-2) through up-regulation of the p53 signaling pathway in CRC mice, which resulted in an adjuvant effect on the treatment of CRC with irinotecan. Moreover, B. longum SX-1326 was also able to regulate the gut-brain-axis (GBA) by restoring damaged enterochromaffin cells, reducing the release of 5-hydroxytryptamine (5-HT) in brain tissue (I vs. U = 89.26 vs. 75.03, p < 0.05), and further alleviating the adverse effects of nausea and vomiting. In addition, B. longum SX-1326 reversed dysbiosis in CRC model mice by increasing the levels of Dehalobacterium, Ruminnococcus, and Mucispirillum. And further alleviated colorectal inflammation by downregulating the TLR4/MyD88/NF-κB signaling pathway. CONCLUSIONS: In conclusion, our work reveals that B. longum SX-1326 has a favorable effect in adjuvant irinotecan for CRC and amelioration of post-chemotherapy side effects, and also provides the theoretical basis and data for finding a safe and efficient chemotherapeutic adjuvant.

Evaluation of In Vivo Prepared Albumin-Drug Conjugate Using Immunoprecipitation Linked LC-MS Assay and Its Application to Mouse Pharmacokinetic Study.

There have been many attempts in pharmaceutical industries and academia to improve the pharmacokinetic characteristics of anti-tumor small-molecule drugs by conjugating them with large molecules, such as monoclonal antibodies, called ADCs. In this context, albumin, one of the most abundant proteins in the blood, has also been proposed as a large molecule to be conjugated with anti-cancer small-molecule drugs. The half-life of albumin is 3 weeks in humans, and its distribution to tumors is higher than in normal tissues. However, few studies have been conducted for the in vivo prepared albumin-drug conjugates, possibly due to the lack of robust bioanalytical methods, which are critical for evaluating the ADME/PK properties of in vivo prepared albumin-drug conjugates. In this study, we developed a bioanalytical method of the albumin-conjugated MAC glucuronide phenol linked SN-38 ((2S,3S,4S,5R,6S)-6-(4-(((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b] quinolin-9-yl)oxy)methyl)(2 (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid) as a proof-of-concept. This method is based on immunoprecipitation using magnetic beads and the quantification of albumin-conjugated drug concentration using LC-qTOF/MS in mouse plasma. Finally, the developed method was applied to the in vivo intravenous (IV) mouse pharmacokinetic study of MAC glucuronide phenol-linked SN-38.

Pogostemon cablin (Blanco) Benth granule revealed a positive effect on improving intestinal barrier function and fecal microbiota in mice with irinotecan-induced intestinal mucositis.

Pogostemon cablin (Blanco) Benth (PCB), a medicinal and edible homologous Chinese herb, has a protective effect on the structure and function of intestine. In this study, we aimed to investigate the effect of PCB granule (PCBG) on the improvement of irinotecan-induced intestinal mucositis and the regulation of intestinal microorganisms in mice. Our results demonstrated that PCBG supplementation significantly improved diarrhea symptoms caused by irinotecan, as evidenced by inhibiting weight loss, reversing intestinal atrophy, protecting against splenomegaly and balancing oxidative stress. Furthermore, compared with the model group, PCBG restored the intestinal morphology and improved intestinal barrier dysfunction by promoting the expression of tight junction proteins and mucin. Moreover, high-throughput sequencing analysis revealed that PCBG improved the flora disorder caused by irinotecan and regulated microbial community structure, such as decreasing the relative abundance of Bacteroides as well as increasing the relative abundance of Lactobacillus. Meanwhile, the disordered microbial functions in intestinal mucositis mice were recovered more closely to the controls by PCBG. Finally, we found that a robust correlation between the specific microbiota and intestinal mucositis-related index. In summary, these findings revealed the beneficial effects of PCBG on the intestinal barrier and gut microbiota of irinotecan-induced intestinal mucositis, which may be one of the potential strategies to reduce the clinical side effects of irinotecan.

Effect of irinotecan administration on amiloride-sensitive sodium taste responses in mice.

Taste alteration is a frequently reported side effect in patients receiving the chemotherapeutic agent, irinotecan. However, the way in which irinotecan causes taste disturbance and the type of taste impairment that is affected remain elusive. Here, we used the two-bottle preference test to characterize behavioral taste responses and employed immunohistochemical analyses to clarify the types and mechanisms of taste alteration induced, in mice, by irinotecan administration. Irinotecan administration resulted in a reduced intake of sodium taste solution but had no effect on sweet taste responses, as determined in the two-bottle preference test. In the presence of amiloride, which inhibits the function of the epithelial sodium channel (ENaC) in the periphery, the intake of sodium taste solution was comparable between the irinotecan-treated and control groups. Immunohistochemical analyses revealed that α-ENaC immunoreactivity detected in taste bud cells decreased slowly after irinotecan administration, and that administration of irinotecan had little effect on the number of cells expressing the cellular proliferation marker, Ki67, within or around taste buds. Our results imply that irinotecan administration may be responsible for altered behavioral sodium taste responses originating from ENaC function in the periphery, while being accompanied by the reduction of α-ENaC expression at the apical membrane of taste receptor cells without disturbing taste cell renewal.

Inhibition of UGT1A1*1 and UGT1A1*6 catalyzed glucuronidation of SN-38 by silybins.

UGT1A1 is the main enzyme that catalyzes the metabolic elimination and detoxification of SN-38, the active form of the drug irinotecan. Milk thistle products have been used widely to protect the liver from injury associated with the use of chemotherapeutic agents. To evaluate whether SN-38 metabolism can be affected by milk thistle products, the inhibitory effects of silybins on UGT1A1*1 and UGT1A1*6 were evaluated in the present investigation. Both silybin A and silybin B potently inhibited SN-38 glucuronidation catalyzed by UGT1A1*1 or UGT1A1*6. It was noteworthy that silybin A and silybin B showed synergistic effect in UGT1A1*1 microsomes at concentration around IC50, while additive effect in UGT1A1*6. According to the predicted AUCi/AUC ratios (the ratio of the area under the plasma concentration-time curve of SN-38 in the presence and absence of silybins), the coadministration of irinotecan and several milk thistle products, including silybin-phosphatidylcholine complex, two Legalon capsules, four Silymarin tablets or four Liverman capsules, may lead to clinically significant herb-drug interactions (HDI) via UGT1A1 inhibition. Meanwhile, Rgut values were much higher than 11 in all the groups, indicating potential HDI due to intestinal UGT1A1 inhibition.

Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating ß­catenin and increases irinotecan sensitivity in colorectal cancer.

With >1.85 million cases and 850,000 deaths annually, colorectal cancer (CRC) is the third most common cancer detected globally. CRC is an aggressive malignancy with metastasis and, in spite of advances in improved treatment regimen, distant disease failure rates remain disappointingly high. Mucin­like 1 (MUCL1) is a small glycoprotein highly expressed mainly in breast cancer. The involvement of the MUCL1 protein in CRC progression and the underlying mechanism have been largely unknown. The aim of the present study was to investigate the MUCL1 expression profile and its functional significance in CRC. The Cancer Genome Atlas dataset revealed that MUCL1 expression was higher in colorectal tumor compared with normal tissues. MUCL1 was also revealed to be expressed in human CRC cell lines. The results demonstrated that MUCL1 promoted cell proliferation and colony formation, confirming its oncogenic potential. Silencing MUCL1 with short interfering RNA inhibited the protein expression of Bcl2 family proteins, such as Bcl2 and BclxL. Targeting MUCL1 resulted in significant inhibition in cell invasive and migratory behavior of HT­29 and SW620 cells. In addition, the expression of E­cadherin increased whereas the expression of vimentin decreased in MUCL1­silenced cells, confirming inhibition of epithelial­mesenchymal transition (EMT) process. Thus, it was revealed that MUCL1 plays a notable role in cell invasion and migration by inhibiting EMT in CRC. Mechanistically, MUCL1 drives ß­catenin activation by Ser­552 phosphorylation, nuclear accumulation and transcriptional activation. Targeting MUCL1 increases the drug sensitivity of CRC cells towards irinotecan. These findings thus demonstrated that MUCL1 acts as a modifier of other pathways that play an important role in CRC progression and MUCL1 was identified as a potential target for CRC therapeutics.

pH-responsive and folate-coated liposomes encapsulating irinotecan as an alternative to improve efficacy of colorectal cancer treatment.

Irinotecan (IRN) is a semisynthetic derivative of camptothecin that acts as a topoisomerase I inhibitor. IRN is used worldwide for the treatment of several types of cancer, including colorectal cancer, however its use can lead to serious adverse effects, as diarrhea and myelosuppression. Liposomes are widely used as drug delivery systems that can improve chemotherapeutic activity and decrease side effects. Liposomes can also be pH-sensitive to release its content preferentially in acidic environments, like tumors, and be surface-functionalized for targeting purposes. Herein, we developed a folate-coated pH-sensitive liposome as a drug delivery system for IRN to reach improved tumor therapy without potential adverse events. Liposomes were prepared containing IRN and characterized for particle size, polydispersity index, zeta potential, concentration, encapsulation, cellular uptake, and release profile. Antitumor activity was investigated in a murine model of colorectal cancer, and its toxicity was evaluated by hematological/biochemical tests and histological analysis of main organs. The results showed vesicles smaller than 200 nm with little dispersion, a surface charge close to neutral, and high encapsulation rate of over 90%. The system demonstrated prolonged and sustained release in pH-dependent manner with high intracellular drug delivery capacity. Importantly, the folate-coated pH-sensitive formulation had significantly better antitumor activity than the pH-dependent system only or the free drug. Tumor tissue of IRN-containing groups presented large areas of necrosis. Furthermore, no evidence of systemic toxicity was found for the groups investigated. Thus, our developed nanodrug IRN delivery system can potentially be an alternative to conventional colorectal cancer treatment.

Synergistic Interactions of Cannabidiol with Chemotherapeutic Drugs in MCF7 Cells: Mode of Interaction and Proteomics Analysis of Mechanisms.

Cannabidiol (CBD), a nonpsychoactive phytocannabinoid, has recently emerged as a potential cytotoxic agent in addition to its ameliorative activity in chemotherapy-associated side effects. In this work, the potential interactions of CBD with docetaxel (DOC), doxorubicin (DOX), paclitaxel (PTX), vinorelbine (VIN), and 7-ethyl-10-hydroxycamptothecin (SN-38) were explored in MCF7 breast adenocarcinoma cells using different synergy quantification models. The apoptotic profiles of MCF7 cells after the treatments were assessed via flow cytometry. The molecular mechanisms of CBD and the most promising combinations were investigated via label-free quantification proteomics. A strong synergy was observed across all synergy models at different molar ratios of CBD in combination with SN-38 and VIN. Intriguingly, synergy was observed for CBD with all chemotherapeutic drugs at a molar ratio of 636:1 in almost all synergy models. However, discording synergy trends warranted the validation of the selected combinations against different models. Enhanced apoptosis was observed for all synergistic CBD combinations compared to monotherapies or negative controls. A shotgun proteomics study highlighted 121 dysregulated proteins in CBD-treated MCF7 cells compared to the negative controls. We reported the inhibition of topoisomerase II ß and α, cullin 1, V-type proton ATPase, and CDK-6 in CBD-treated MCF7 cells for the first time as additional cytotoxic mechanisms of CBD, alongside sabotaged energy production and reduced mitochondrial translation. We observed 91 significantly dysregulated proteins in MCF7 cells treated with the synergistic combination of CBD with SN-38 (CSN-38), compared to the monotherapies. Regulation of telomerase, cell cycle, topoisomerase I, EGFR1, protein metabolism, TP53 regulation of DNA repair, death receptor signalling, and RHO GTPase signalling pathways contributed to the proteome-wide synergistic molecular mechanisms of CSN-38. In conclusion, we identified significant synergistic interactions between CBD and the five important chemotherapeutic drugs and the key molecular pathways of CBD and its synergistic combination with SN-38 in MCF7 cells. Further in vivo and clinical studies are warranted to evaluate the implementation of CBD-based synergistic adjuvant therapies for breast cancer.

Heterogenous chemosensitivity of a panel of organoid lines derived from small cell neuroendocrine carcinoma of the uterine cervix.

Small cell neuroendocrine carcinoma (SCNEC) of the uterine cervix is a rare disease with a poor prognosis. The lack of established disease models has hampered therapy development. We generated a panel of cancer tissue-originated spheroid (CTOS) lines derived from SCNEC of the uterine cervix using a method based upon cell-cell contact throughout the preparation and culturing processes. Using 11 CTOS lines, we assessed the sensitivity of various drugs used in clinical practice. Drug sensitivity assays revealed significant heterogeneous inter-CTOS chemosensitivity. Microarray analyses were then performed to identify sensitivity-related gene signatures. Specific gene sets were identified which likely contribute to the sensitivity to the tested drugs. We identified a line (Cerv54) that was exceptionally sensitive to irinotecan. Cerv54 had increased levels of CES1, which catalyzes the conversion of irinotecan to the active form, SN38, although in Cerv54 cells, SN38 was undetectable, CES1 expression and activity were markedly low compared to the liver, and a CES1 inhibitor had no effect on irinotecan sensitivity. These results suggested a novel irinotecan mode of action in Cerv54. Our CTOS lines may be useful for understanding the variation and mechanism of drug sensitivity, contributing to the understanding and development of chemotherapeutic drugs.

The transport pathway in the ABCG2 protein and its regulation revealed by molecular dynamics simulations.

Atomic-level structural insight on the human ABCG2 membrane protein, a pharmacologically important transporter, has been recently revealed by several key papers. In spite of the wealth of structural data, the pathway of transmembrane movement for the large variety of structurally different ABCG2 substrates and the physiological lipid regulation of the transporter has not been elucidated. The complex molecular dynamics simulations presented here may provide a breakthrough in understanding the steps of the substrate transport process and its regulation by cholesterol. Our analysis revealed drug binding cavities other than the central binding site and delineated a putative dynamic transport pathway for substrates with variable structures. We found that membrane cholesterol accelerated drug transport by promoting the closure of cytoplasmic protein regions. Since ABCG2 is present in all major biological barriers and drug-metabolizing organs, influences the pharmacokinetics of numerous clinically applied drugs, and plays a key role in uric acid extrusion, this information may significantly promote a reliable prediction of clinically important substrate characteristics and drug-drug interactions.

A PEGylated alternating copolymeric prodrug of sulfur dioxide with glutathione responsiveness for Irinotecan delivery.

In this study, an enhanced anticancer strategy combining the chemotherapy from antineoplastics with the oxidative damage from a sulfur dioxide (SO2) prodrug is presented. Based on the characteristics of a high glutathione (GSH) level in the tumor microenvironment, a novel GSH-responsive SO2 polymeric prodrug mPEG-b-P(PA-alt-GDNs) was designed and synthesized via a ring-opening alternating copolymerization and "click" reaction. The GSH-sensitive mechanism of the polymer was investigated in detail. Furthermore, Irinotecan was loaded into the polymeric prodrug nanoparticles by a self-assembly method with a drug loading content of 12.3 wt% and a loading efficiency of 42.2%. The drug-loaded nanoparticles showed a sensitive response to high concentrations of GSH in the tumor cells and rapidly released both Irinotecan and SO2. The depletion of GSH and the release of SO2 were supposed to increase the level of reactive oxygen species in the tumor cell, which, in combination with the released Irinotecan, exerted an enhanced anti-proliferative effect against HepG2 cells. Finally, Irinotecan-loaded nanoparticles exhibited a stronger antitumor effect than free antineoplastics in HepG2 cells. Thus, these results indicated that our polymeric prodrug SO2 is a promising candidate for chemotherapeutic drug delivery and would be a new weapon in anticancer treatment.

Significance of aldo-keto reductase 1C3 and ATP-binding cassette transporter B1 in gain of irinotecan resistance in colon cancer cells.

Irinotecan (CPT11) is widely prescribed for treatment of various intractable cancers such as advanced and metastatic colon cancer cells, but its continuous treatment promotes the resistance development. In this study, we established CPT11-resistant variants of three human colon cancer (DLD1, RKO and LoVo) cell lines, and found that gain of the resistance elicited an up-regulation of aldo-keto reductase (AKR) 1C3 in the cells. Additionally, the sensitivity to CPT11 toxicity was decreased and increased by overexpression and knockdown, respectively, of the enzyme. Moreover, the resistant cells suppressed formation of reactive 4-hydroxy-2-nonenal by CPT11 treatment, and the suppressive effect was almost completely abolished by addition of an AKR1C3 inhibitor. These results suggest that up-regulated AKR1C3 contributes to promotion of the chemoresistance by detoxifying the reactive aldehyde. Western blot and real-time polymerase-chain reaction analyses and ATP-binding cassette (ABC) B1-functional assay revealed that, among three ABC transporters, ABCB1 was the most highly up-regulated by development of the CPT11 resistance, inferring a significant contribution of pregnane-X receptor-dependent signaling to the ABCB1 up-regulation. The combined treatment with inhibitors of AKR1C3 and ABCB1 potently sensitized the resistant cells to CPT11 and its active metabolite SN38. Taken together, our results suggest that combination of AKR1C3 and ABCB1 inhibitors is effective as adjuvant therapy to enhance CPT11 sensitivity of intractable colon cancer cells.

Combinational dual drug delivery system to enhance the care and treatment of gastric cancer patients.

Gastric cancer is a frequently occurring cancer with high mortality each year worldwide. Finding new and effective therapeutic strategy against human gastric cancer is still urgently required. Hence, we have established a new method to achieve treatment-actuated modifications in a tumor microenvironment by utilizing synergistic activity between two potential anticancer drugs. Dual drug delivery of gemcitabine (GEM) and Camptothecin-11 (CPT-11) exhibits a great anti-cancer potential, as GEM enhances the effect of CPT-11 treatment of human gastric cells by providing microenvironment stability. However, encapsulation of GEM and CPT-11 obsessed by poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) is incompetent owing to unsuitability between the binary free GEM and CPT-11 moieties and the polymeric system. Now, we display that CPT-11 can be prepared by hydrophobic covering of the drug centers with dioleoylphosphatidic acid (DOPA). The DOPA-covered CPT-11 can be co-encapsulated in PLGA NPs alongside GEM to stimulate excellent anticancer property. The occurrence of the CPT-11 suggestively enhanced the encapsulations of GEM into PLGA NPs (GEM-CPT-11 NPs). Formation of the nanocomposite (GEM-CPT-11 NPs) was confirmed by FTIR and X-ray spectroscopic techniques. Further, the morphology of GEM NPs, CPT-11 NPs, and GEM-CPT-11 NPs and NP size was examined by transmission electron microscopy (TEM), respectively. Furthermore, GEM-CPT-11 NPs induced significant apoptosis in human gastric NCI-N87 and SGC-791 cancer cells in vitro. The morphological observation and apoptosis were confirmed by the various biochemical assays (AO-EB, nuclear staining, and annexin V-FITC). In addition, evaluation of the hemolysis assay with erythrocytes of human shows excellent biocompatibility of free GEM, free CPT-11, GEM NPs, CPT-11 NPs, and GEM-CPT-11 NPs. The results suggest that GEM-CPT-11 NPs are one of the promising nursing cares for human gastric cancer therapeutic candidates worthy of further investigations.

Effect of drug metabolizing enzymes and transporters in Thai colorectal cancer patients treated with irinotecan-based chemotherapy.

Genetic polymorphisms in drug metabolizing enzymes and drug transporters may affect irinotecan toxicity. Although genetic polymorphisms have been shown to influence the irinotecan toxicity, data are limited in Thai population. Thus, the aim of this study was to assess the allele and genotype frequencies and the relationship between CYP3A4/5, DPYD, UGT1A1, ABCB1, and ABCC2 genetic variations and irinotecan-induced toxicity in Thai colorectal cancer patients. One hundred and thirty-two patients were genotyped, and the effect of genetic variations on irinotecan-induced toxicity was assessed in 66 patients who received irinotecan-based chemotherapy. Allele frequencies of ABCB1 c.1236C > T, ABCB1 c.3435C > T, ABCC2 c.3972C > T, ABCG2 c.421C > A, CYP3A4*1B, CYP3A4*18, CYP3A5*3, DPYD*5, UGT1A1*28, and UGT1A1*6 were 0.67, 0.43, 0.23, 0.27, 0.01, 0.02, 0.64, 0.19, 0.16, and 0.09, respectively. DPYD*2A and DPYD c.1774C > T variants were not detected in our study population. The ABCC2 c.3972C > T was significantly associated with grade 1-4 neutropenia (P < 0.012) at the first cycle. Patients carrying both UGT1A1*28 and *6 were significantly associated with severe neutropenia at the first (P < 0.001) and second (P = 0.017) cycles. In addition, patients carrying UG1A1*28 and *6 had significantly lower absolute neutrophil count (ANC) nadir at first (P < 0.001) and second (P = 0.001) cycles. This finding suggests that UGT1A1*28, *6, and ABCC2 c.3972C > T might be an important predictor for irinotecan-induced severe neutropenia.

CTDSP1 inhibitor rabeprazole regulates DNA-PKcs dependent topoisomerase I degradation and irinotecan drug resistance in colorectal cancer.

Irinotecan specifically targets topoisomerase I (topoI), and is used to treat various solid tumors, but only 13-32% of patients respond to the therapy. Now, it is understood that the rapid rate of topoI degradation in response to irinotecan causes irinotecan resistance. We have published that the deregulated DNA-PKcs kinase cascade ensures rapid degradation of topoI and is at the core of the drug resistance mechanism of topoI inhibitors, including irinotecan. We also identified CTD small phosphatase 1 (CTDSP1) (a nuclear phosphatase) as a primary upstream regulator of DNA-PKcs in response to topoI inhibitors. Previous reports showed that rabeprazole, a proton pump inhibitor (PPI) inhibits CTDSP1 activity. The purpose of this study was to confirm the effects of rabeprazole on CTDSP1 activity and its impact on irinotecan-based therapy in colon cancer. Using differentially expressing CTDSP1 cells, we demonstrated that CTDSP1 contributes to the irinotecan sensitivity by preventing topoI degradation. Retrospective analysis of patients receiving irinotecan with or without rabeprazole has shown the effects of CTDSP1 on irinotecan response. These results indicate that CTDSP1 promotes sensitivity to irinotecan and rabeprazole prevents this effect, resulting in drug resistance. To ensure the best chance at effective treatment, rabeprazole may not be a suitable PPI for cancer patients treated with irinotecan.

Design, Synthesis, and Biological Evaluation of HSP90 Inhibitor-SN38 Conjugates for Targeted Drug Accumulation.

Herein, a series of HSP90 inhibitor-SN38 conjugates through ester and carbamate linkage in the 20-OH and 10-OH positions of SN38 were developed for improving the tumor-specific penetration and accumulation of SN38 via extracellular HSP90 (eHSP90)-mediated endocytosis. Mechanistic analyses confirmed that these novel conjugates could bind to eHSP90 and be selectively internalized into the tumor cells, which led to prolonged tumor regression in multiple models of cancer. Among all studied conjugates, compound 18b showed excellent in vitro activities, including acceptable HSP90α affinity and potent antitumor activity. Moreover, compound 18b exhibited superior antitumor activity and low toxicity in HCT116 and Capan-1 xenograft models. Pharmacokinetic analyses in HCT116 and Capan-1 xenografts further confirmed that compound 18b treatment could lead to effective cleavage and extended SN38 exposure at tumor sites. All these encouraging data indicate that this compound is a promising new candidate for cancer therapy and merits further chemical and biological evaluation.

Enhanced cellular uptake efficiency of DCM probes or SN38 conjugating with phenylboronic acids.

High-level of sialic acid (SA) expression on the surface of cancer cells is observed extremely common. Phenylboronic acids (PBAs) have a high affinity with SA. The cellular uptake efficiency could be enhanced by the strategy of introducing PBA fragments to the compounds. In this work, we synthesized five new probes with the Dicyanomethylene-4H-pyran (DCM) fluorophore, three of them conjugated with different phenylboronic acid fragments. By cellular uptake experiments, DLCB and DLAB showed enhanced cellular uptake abilities compared with DLN and DLO. These two effective phenylboronic acid fragments were then conjugated with SN-38 and the conjugates showed enhanced cellular uptake abilities by 3-fold or 7-fold compared with irinotecan. In summary, the strategy of introducing 4-carboxyphenylboronic acid and 3-amino-benzoxaborole groups shows great potential in drug delivery system. Moreover, the released linkers between boric acid and drugs deserve further studies.

Regulation of breast cancer resistance protein and P-glycoprotein by ezrin, radixin and moesin in lung, intestinal and renal cancer cell lines.

OBJECTIVES: Ezrin (Ezr), radixin (Rdx) and moesin (Msn) (ERM) proteins anchor other proteins to the cell membrane, serving to regulate their localization and function. Here, we examined whether ERM proteins functionally regulate breast cancer resistance protein (BCRP) and P-glycoprotein in cell lines derived from lung, intestinal and renal cancers. METHODS: ERM proteins were each silenced with appropriate siRNA. BCRP and P-gp functions were evaluated by means of efflux and uptake assays using 7-ethyl-10-hydroxycamptothecin (SN-38) and rhodamine123 (Rho123) as specific substrates, respectively, in non-small cell lung cancer HCC827 cells, intestinal cancer Caco-2 cells and renal cancer Caki-1 cells. KEY FINDINGS: In HCC827 cells, the efflux rates of SN-38 and Rho123 were significantly decreased by knockdown of Ezr or Msn, but not Rdx. However, BCRP function was unaffected by Ezr or Rdx knockdown in Caco-2 cells, which do not express Msn. In Caki-1 cells, Rdx knockdown increased the intracellular SN-38 concentration, while knockdown of Ezr or Msn had no effect. CONCLUSIONS: Our findings indicate that regulation of BCRP and P-gp functions by ERM proteins is organ-specific. Thus, if the appropriate ERM protein(s) are functionally suppressed, accumulation of BCRP or P-gp substrates in lung, intestine or kidney cancer tissue might be specifically increased.

Sacituzumab govitecan, an antibody-drug conjugate targeting trophoblast cell-surface antigen 2, shows cytotoxic activity against poorly differentiated endometrial adenocarcinomas in vitro and in vivo.

Endometrial cancer is the most common gynecologic malignancy in developed countries. The antibody-drug conjugate (ADC) sacituzumab govitecan (SG) targets trophoblast cell-surface antigen-2 (Trop-2) - a cell-surface glycoprotein highly expressed in many epithelial tumors - and delivers the active metabolite of irinotecan SN-38 to Trop-2-positive tumor cells. We evaluated Trop-2 expression in endometrial endometrioid carcinoma (EC) tissues and the activity of SG against primary poorly differentiated EC cell lines and xenografts. Trop-2 expression was assessed in 143 formalin-fixed-paraffin-embedded tumors and seven primary tumor cell lines by immunohistochemistry and flow cytometry, respectively. Cell viability of primary tumor cell lines was assessed following exposure to SG, or control antibodies. Antibody-dependent cell cytotoxicity (ADCC) against Trop-2-positive and Trop-2-negative EC cell lines was measured in vitro using 4-h chromium release assays. A Trop-2-positive EC xenograft model was used to determine the in vivo activity of SG. Moderate-to-strong staining was detected in 84% (120/143) of EC samples, whereas 43% (3/7) of the primary EC cell lines tested overexpressed Trop-2. EC cell lines overexpressing Trop-2 were significantly more sensitive to SG compared to control ADC (P = 0.014 and P = 0.005). Both SG and the unconjugated parental antibody hRS7 mediated high ADCC against Trop-2-positive cell lines. Moreover, SG induced significant bystander killing of Trop-2-negative tumors cocultured with Trop-2-positive tumors. In the xenograft model, intravenous administration of SG twice weekly for three weeks was well tolerated and demonstrated impressive tumor growth inhibition against poorly differentiated, chemotherapy-resistant EC xenografts (P = 0.011). In summary, SG is a novel ADC with remarkable preclinical activity against poorly differentiated EC cell lines overexpressing Trop-2. These findings warrant future clinical trials.

The Emerging Role of the SLCO1B3 Protein in Cancer Resistance.

Currently, chemotherapy is one of the mainstays of oncologic therapies. But the efficacy of chemotherapy is often limited by drug resistance and severe side effects. Consequently, it is becoming increasingly important to investigate the underlying mechanism and overcome the problem of anticancer chemotherapy resistance. The solute carrier organic anion transporter family member 1B3 (SLCO1B3), a functional transporter normally expressed in the liver, transports a variety of endogenous and exogenous compounds, including hormones and their conjugates as well as some anticancer drugs. The extrahepatic expression of SLCO1B3 has been detected in different cancer cell lines and cancer tissues. Recently, accumulating data indicates that the abnormal expression and function of SLCO1B3 are involved in resistance to anticancer drugs, such as taxanes, camptothecin and its analogs, SN-38, and Androgen Deprivation Therapy (ADT) in breast, prostate, lung, hepatic, and colorectal cancer, respectively. Thus, more investigations have been implemented to identify the potential SLCO1B3-related mechanisms of cancer drug resistance. In this review, we focus on the emerging roles of SLCO1B3 protein in the development of cancer chemotherapy resistance and briefly discuss the mechanisms of resistance. Elucidating the function of SLCO1B3 in chemoresistance may bring out novel therapeutic strategies for cancer treatment.