Design, synthesis and SAR study of 3rd-generation taxoids bearing 3-CH3, 3-CF3O and 3-CHF2O groups at the C2-benzoate position.

It has been shown that inclusion of CF3O and CHF2O groups to drug candidates often improve their pharmacological properties, especially metabolic stability, membrane permeability and PK profile. Moreover, the unique non-spherical structure of the OCHF2 group can provide interesting and beneficial characteristics. Accordingly, new 3rd-generation taxoids, bearing 3-OCF3 or 3-OCF2H (and 3-CH3 for comparison) at the C2 benzoate moiety, were synthesized and their potencies against drug-sensitive and drug-resistant cancer cell lines examined. In this study, our previous SAR studies on 3rd-generation taxoids were expanded to disclose that CH3, CF3O and CHF2O groups are well tolerated at this position and enhance potency, especially against MDR-cancer cell lines so that these taxoids can virtually overcome MDR. These new taxoids exhibit up to 7 times higher cytotoxicity (IC50) than paclitaxel against drug-sensitive cancer cell lines (MCF7 and LCC6-WT) and 2-3 orders of magnitude higher potency than paclitaxel against drug-resistant ovarian, breast and colon cancer cell lines with MDR-phenotype (NCI/ADR, LCC6-MDR and LDL-1), as well as pancreatic cancer cell line, CFPAC-1. Since it has been shown that a bulky group at this position reduces potency, it is noteworthy that rather bulky CF3O and CHF2O groups are well tolerated. Molecular modeling analysis indicated the favorable van der Waals interactions of CF3O and CHF2O groups in the binding site. It is also worthy of note that new taxoids, bearing a CHF2O group at the C2 benzoate position (1-06 series), exhibited the highest potencies against MDR-cancer cell lines and cancer stem cell (CSC)-enriched cancer cell lines. These new 3rd-generation taxoids are promising candidates for highly potent chemotherapeutic agents, as well as payloads for tumor-targeting drug conjugates such as antibody-drug conjugates (ADCs).

Biodistribution and Pharmacokinetic Evaluations of a Novel Taxoid DHA-SBT-1214 in an Oil-in-Water Nanoemulsion Formulation in Naïve and Tumor-Bearing Mice.

PURPOSE: The main purpose of this study was to formulate an oil-in-water nanoemulsion of a next generation taxoid DHA-SBT-1214 and evaluate its biodistribution and pharmacokinetics. METHODS: DHA-SBT-1214 was encapsulated in a fish oil containing nanoemulsion using a high pressure homogenization method. Following morphological characterization of the nanoemulsions, qualitative and quantitative biodistribution was evaluated in naïve and cancer stem cell-enriched PPT-2 human prostate tumor bearing mice. RESULTS: DHA-SBT-1214 was successfully encapsulated up to 20 mg/ml in the nanoemulsion formulation and had an average oil droplet size of 200 nm. Using a DiR near infra-red dye encapsulated nanoemulsion, we have shown the delivery of nanoemulsion to mouse tumor region. By quantitative analysis, DHA-SBT-1214 encapsulated nanoemulsion demonstrated improved pharmacokinetic properties in plasma and different tissues as compared to its solution form. Furthermore, the nanoemulsions were stable and had slower in vitro drug release compared to its solution form. CONCLUSIONS: The results from this study demonstrated effective encapsulation of the drug in a nanoemulsion and this nanoemulsion showed sustained plasma levels and enhanced tumor delivery relative to the solution form.

Nanoemulsion formulation of a novel taxoid DHA-SBT-1214 inhibits prostate cancer stem cell-induced tumor growth.

The main aim of this study was to evaluate the therapeutic efficacy of an oil-in-water nanoemulsion formulation encapsulating DHA-SBT-1214, a novel omega-3 fatty acid conjugated taxoid prodrug, against prostate cancer stem cells. Nanoemulsions of DHA-SBT-1214 (NE-DHA-SBT-1214) were prepared and characterized. In vitro delivery efficiency and cytotoxicity of NE-DHA-SBT-1214 was compared with solution formulation in PPT2 cells. In vivo studies included analysis of comparative efficacy of NE-DHA-SBT-1214 with Abraxane® and placebo nanoemulsions as well as post-treatment alternations in clonogenic and sphere-forming capabilities of the tumor cells. Qualitative intracellular uptake studies of dye encapsulated NEs by confocal imaging showed uptake by both monolayer and spheroid cultured PPT2 cells. Treatment of PPT2 cells with NE DHA-SBT-1214 (1nM-1µM for monolayer culture of cells grown on collagen-coated dishes for 48 h) induced complete cell death, showing higher efficacy as compared to the drug solution. This nanoemulsion (10nM-10µM) also showed toxicity in 3D culture of floating spheroids. Weekly intravenous administration of the NE-DHA-SBT-1214 to NOD/SCID mice bearing subcutaneous PPT2 tumor xenografts led to dramatic suppression of tumor growth compared to Abraxane® and placebo nanoemulsion formulation. Viable cells that survived from this in vivo treatment regimen were no longer able to induce floating spheroids and holoclones, whereas control and Abraxane® treated tumor cells induced a large number of both. The results show that NE-DHA-SBT-1214 possesses significant activity against prostate CD133high/CD44+/high tumor-initiating cells both in vitro and in vivo.

Establishment of highly tumorigenic human colorectal cancer cell line (CR4) with properties of putative cancer stem cells.

BACKGROUND: Colorectal cancer (CRC) has the third highest mortality rates among the US population. According to the most recent concept of carcinogenesis, human tumors are organized hierarchically, and the top of it is occupied by malignant stem cells (cancer stem cells, CSCs, or cancer-initiating cells, CICs), which possess unlimited self-renewal and tumor-initiating capacities and high resistance to conventional therapies. To reflect the complexity and diversity of human tumors and to provide clinically and physiologically relevant cancer models, large banks of characterized patient-derived low-passage cell lines, and especially CIC-enriched cell lines, are urgently needed. PRINCIPAL FINDINGS: Here we report the establishment of a novel CIC-enriched, highly tumorigenic and clonogenic colon cancer cell line, CR4, derived from liver metastasis. This stable cell line was established by combining 3D culturing and 2D culturing in stem cell media, subcloning of cells with particular morphology, co-culture with carcinoma associated fibroblasts (CAFs) and serial transplantation to NOD/SCID mice. Using RNA-Seq complete transcriptome profiling of the tumorigenic fraction of the CR4 cells in comparison to the bulk tumor cells, we have identified about 360 differentially expressed transcripts, many of which represent stemness, pluripotency and resistance to treatment. Majority of the established CR4 cells express common markers of stemness, including CD133, CD44, CD166, EpCAM, CD24 and Lgr5. Using immunocytochemical, FACS and western blot analyses, we have shown that a significant ratio of the CR4 cells express key markers of pluripotency markers, including Sox-2, Oct3/4 and c-Myc. Constitutive overactivation of ABC transporters and NF-kB and absence of tumor suppressors p53 and p21 may partially explain exceptional drug resistance of the CR4 cells. CONCLUSIONS: The highly tumorigenic and clonogenic CIC-enriched CR4 cell line may provide an important new tool to support the discovery of novel diagnostic and/or prognostic biomarkers as well as the development of more effective therapeutic strategies.

Prostate cancer stem cell-targeted efficacy of a new-generation taxoid, SBT-1214 and novel polyenolic zinc-binding curcuminoid, CMC2.24.

BACKGROUND: Prostate cancer is the second leading cause of cancer death among men. Multiple evidence suggests that a population of tumor-initiating, or cancer stem cells (CSCs) is responsible for cancer development and exceptional drug resistance, representing a highly important therapeutic target. The present study evaluated CSC-specific alterations induced by new-generation taxoid SBT-1214 and a novel polyenolic zinc-binding curcuminoid, CMC2.24, in prostate CSCs. PRINCIPAL FINDINGS: The CD133(high)/CD44(high) phenotype was isolated from spontaneously immortalized patient-derived PPT2 cells and highly metastatic PC3MM2 cells. Weekly treatment of the NOD/SCID mice bearing PPT2- and PC3MM3-induced tumors with the SBT-1214 led to dramatic suppression of tumor growth. Four of six PPT2 and 3 of 6 PC3MM2 tumors have shown the absence of viable cells in residual tumors. In vitro, SBT-1214 (100 nM-1 µM; for 72 hr) induced about 60% cell death in CD133(high)/CD44(+/high) cells cultured on collagen I in stem cell medium (in contrast, the same doses of paclitaxel increased proliferation of these cells). The cytotoxic effects were increased when SBT-1214 was combined with the CMC2.24. A stem cell-specific PCR array assay revealed that this drug combination mediated massive inhibition of multiple constitutively up-regulated stem cell-related genes, including key pluripotency transcription factors. Importantly, this drug combination induced expression of p21 and p53, which were absent in CD133(high)/CD44(high) cells. Viable cells that survived this treatment regimen were no longer able to induce secondary spheroids, exhibited significant morphological abnormalities and died in 2-5 days. CONCLUSIONS: We report here that the SBT-1214 alone, or in combination with CMC2.24, possesses significant activity against prostate CD133(high)/CD44(+/high) tumor-initiating cells. This drug combination efficiently inhibits expression of the majority of stem cell-related genes and pluripotency transcription factors. In addition, it induces a previously absent expression of p21 and p53 ("gene wake-up"), which can potentially reverse drug resistance by increasing sensitivity to anti-cancer drugs.

Colon cancer stem cells--from basic to clinical application.

Based on cancer stem cell (CSC) concept of carcinogenesis, tumors represent complex heterogeneous organ-like systems with a hierarchical cellular organization, and only minority phenotypic subpopulations with stem-like properties possess a dual ability to self-renew indefinitely and produce all the heterogeneous cell phenotypes comprising the bulk tumor cells. Large experimental and clinical data indicate that conventional anti-cancer therapies cannot eradicate CSCs, and moreover, they usually increase their number leading to cancer recurrence and further drug resistance. In this review, several current controversies in the CSC field and recent studies, which help to shed light on their origin, are discussed. The emerging necessity for the development of complex, multimodal CSC-targeted treatment strategies, which combine conventional therapeutics with promising pathway-specific modulators, and natural compounds, which can improve the efficacy of conventional anti-cancer therapeutics and decrease their undesirable side effects is presented. Also, novel requirements and criteria necessary for evaluation of the CSC-targeted drug efficacy and relevant experimental models are discussed.

New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheroids induced by purified colon tumor-initiating cells.

BACKGROUND: Growing evidence suggests that the majority of tumors are organized hierarchically, comprising a population of tumor-initiating, or cancer stem cells (CSCs) responsible for tumor development, maintenance and resistance to drugs. Previously we have shown that the CD133high/CD44high fraction of colon cancer cells is different from their bulk counterparts at the functional, morphological and genomic levels. In contrast to the majority of colon cancer cells expressing moderate levels of CD133, CD44 and CD166, cells with a high combined expression of CD133 and CD44 possessed several characteristic stem cell features, including profound self-renewal capacity in vivo and in vitro, and the ability to give rise to different cell phenotypes. The present study was undertaken for two aims: a) to determine stem cell-related genomic characteristics of floating 3D multicellular spheroids induced by CD133high/CD44high colon cancer cells; and b) to evaluate CSC-specific alterations induced by new-generation taxoid SB-T-1214. RESULTS: Selected CSC phenotype was isolated from three independent invasive colon cancer cell lines, HCT116, HT29 and DLD-1. A stem cell-specific PCR array assay (SABiosciences) revealed that colonospheres induced by purified CD133high/CD44high expressing cells display profound up-regulation of stem cell-related genes in comparison with their bulk counterparts. The FACS analysis has shown that the 3D colonospheres contained some minority cell populations with high levels of expression of Oct4, Sox2, Nanog and c-Myc, which are essential for stem cell pluripotency and self-renewal. Single administration of the SB-T-1214 at concentration 100 nM-1 microM for 48 hr not only induced growth inhibition and apoptotic cell death in these three types of colon cancer spheroids in 3D culture, but also mediated massive inhibition of the stem cell-related genes and significant down-regulation of the pluripotency gene expression. PCR array and FACS data were confirmed with western blotting. Importantly, viable cells that survived this treatment regimen were no longer able to induce secondary floating spheroids and exhibited significant morphological abnormalities. CONCLUSIONS: We report here that a new-generation taxoid SB-T-1214 possesses significant activity against colon cancer spheroids induced by and enriched with drug resistant tumorigenic CD133high/CD44high cells and efficiently inhibited expression of the majority of stem cell-related genes. Our data indicates that the previously observed long-term efficacy of SB-T-1214 against drug resistant colon tumors in vivo may be explained by the down-regulation of multiple stem cell-related genes in the tumorigenic cell population, in addition to its known efficacy as a mitotic poison against proliferating cancer cells.

Molecular mechanism of chemoresistance by miR-215 in osteosarcoma and colon cancer cells.

BACKGROUND: Translational control mediated by non-coding microRNAs (miRNAs) plays a key role in the mechanism of cellular resistance to anti-cancer drug treatment. Dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS, TS) are two of the most important targets for antifolate- and fluoropyrimidine-based chemotherapies in the past 50 years. In this study, we investigated the roles of miR-215 in the chemoresistance to DHFR inhibitor methotrexate (MTX) and TS inhibitor Tomudex (TDX). RESULTS: The protein levels of both DHFR and TS were suppressed by miR-215 without the alteration of the target mRNA transcript levels. Interestingly, despite the down-regulation of DHFR and TS proteins, ectopic expression of miR-215 resulted in a decreased sensitivity to MTX and TDX. Paradoxically, gene-specific small-interfering RNAs (siRNAs) against DHFR or TS had the opposite effect, increasing sensitivity to MTX and TDX. Further studies revealed that over-expression of miR-215 inhibited cell proliferation and triggered cell cycle arrest at G2 phase, and that this effect was accompanied by a p53-dependent up-regulation of p21. The inhibitory effect on cell proliferation was more pronounced in cell lines containing wild-type p53, but was not seen in cells transfected with siRNAs against DHFR or TS. Moreover, denticleless protein homolog (DTL), a cell cycle-regulated nuclear and centrosome protein, was confirmed to be one of the critical targets of miR-215, and knock-down of DTL by siRNA resulted in enhanced G2-arrest, p53 and p21 induction, and reduced cell proliferation. Additionally, cells subjected to siRNA against DTL exhibited increased chemoresistance to MTX and TDX. Endogenous miR-215 was elevated about 3-fold in CD133+HI/CD44+HI colon cancer stem cells that exhibit slow proliferating rate and chemoresistance compared to control bulk CD133+/CD44+ colon cancer cells. CONCLUSIONS: Taken together, our results indicate that miR-215, through the suppression of DTL expression, induces a decreased cell proliferation by causing G2-arrest, thereby leading to an increase in chemoresistance to MTX and TDX. The findings of this study suggest that miR-215 may play a significant role in the mechanism of tumor chemoresistance and it may have a unique potential as a novel biomarker candidate.

Phenotypic subpopulations of metastatic colon cancer stem cells: genomic analysis.

BACKGROUND: Human cancer is characterized by high heterogeneity in gene expression, varieties of differentiation phenotypes and tumor-host interrelations. Growing evidence suggests that tumor-initiating, or cancer stem cells (CSCs), may also represent a heterogeneous population. The present study was undertaken to isolate and characterize the different phenotypic subpopulations of metastatic colon cancer and to develop a working colon CSC model for obtaining highly tumorigenic and clonogenic cells in sufficient numbers. MATERIALS AND METHODS: Different phenotypic cell subpopulations were isolated based on differential levels and patterns of expression of several stemness markers, including CD133, CD44, CD166 and CD49b. Stemness properties of isolated cells were tested by analysis of their ability to form floating colonospheres in vitro, to induce tumors in NOD/SCID mice after transplantation at relatively low cell numbers, and to produce progenitors of different phenotypes. RESULTS: The metastatic colon cancer HCT116 cell line, which expressed a majority of known CSC markers, closely resembling the patterns of expression in exfoliated peritoneal cells from several metastatic colon cancer patients, was selected as a reference material. Genome-wide microarray analysis (Affymetrix; DAVID) of CD133(high) CSC-enriched versus CSC-depleted cell populations revealed 4,351 differentially expressed genes with an overrepresentation of those responsible for apoptosis resistance, regulation of cell cycle, proliferation, stemness and developmental pathways. Simultaneous analysis of 84 stem cell- and metastasis-related genes with corresponding PCR arrays identified genes differentially expressed in several colon CSC phenotypic populations versus bulk tumor cells, and in relation to each other. It was found that colonospheres induced by tumorigenic cells with the highest expression of CD133 and those which were induced by CD133/CD44-negative cells possessed profoundly different stem cell-related gene expression profiles. CONCLUSION: The proposed approaches allow for reliable isolation and propagation of highly tumorigenic and clonogenic cells of different phenotypes. Genomic analysis of several candidate CSC phenotypic populations may contribute to the identification of novel targets for colon cancer stem cell-targeted drug development and treatment.

Genomic analysis of prostate cancer stem cells isolated from a highly metastatic cell line.

BACKGROUND: Tumor-initiating or cancer stem cells (CSCs) were recently isolated from all major human cancers, including prostate cancer. However, the extreme heterogeneity of tumor cells in terms of biological behavior and gene expression patterns and difficulties isolating a pure population of CSCs from tumor tissues significantly impede a comparative analysis of CSCs. MATERIALS AND METHODS: Different phenotypic populations were isolated from a metastatic derivative of PC-3 cell line, PC3-MM2, and tested for their ability to form tumors in NOD/SCID mice and floating spheroids in 3D culture systems. RESULTS: All tested cell lines possessed minor populations of cells with highest expression of CD133, CD44 and CD166, whereas the vast majority of cells were CD133-negative. Several experimental approaches promoted a higher proportion of CD133-positive cells with increased in vivo tumorigenicity and the ability to produce floating spheres. Genome-wide microarray analysis (Affymetrix; DAVID) of CSC-enriched versus CSC-depleted cell populations revealed 213 genes with 10-100 fold increased activity out of 8994 differentially expressed ones and 87 genes with 5-50 fold decreased activity. CONCLUSIONS: The proposed in vitro prostate CSC model allows for reliable isolation and propagation of highly tumorigenic cells. This study may contribute to the identification of novel targets for CSC-targeted prostate cancer treatment.

Clinical significance of telomerase activity in peritoneal disseminated cells: gastrointestinal cancers.

Early detection and accurate staging of gastrointestinal (GI) cancers are difficult. The aim of this study was to evaluate whether telomerase activity (TA) in exfoliated/disseminated epithelial cells could be used as a reliable marker for GI cancers. TA was evaluated with the real-time RTQ-TRAP in immunomagnetically sorted peritoneal epithelial cells from 60 patients undergoing surgical treatment. Thirty-two patients were clinically diagnosed with a variety of GI cancers: 1 had premalignant disease, 2 had history of GI cancers, and 25 patients were clinically negative for cancer. Here we report that all types and all cases of gastrointestinal cancers were telomerase positive, thereby demonstrating 100% sensitivity for cancer. Eighteen of 25 nonmalignant cases had undetectable levels of TA, 2 had low, and 5 of 25 expressed high TA levels. Because normal epithelial cells usually have low TA and a lesser tendency to exfoliate compared with cancer cells, it is of great importance to have close follow-up for these patients to exclude possible malignant disease. We conclude that RTQ-TRAP assessment of TA in immunomagnetically sorted peritoneal epithelial cells has 100% sensitivity and 100% negative predictive value for GI cancers, and therefore, can be considered as a valuable tool and useful addition to current standard diagnostic methods. Clinical significance of unusually high telomerase activity in some clinically negative for cancer cases requires further study.

Highly sensitive revealing of PCR products with capillary electrophoresis based on single photon detection.

Post-PCR fragment analysis was conducted using our single photon detection-based DNA sequencing instrument in order to substantially enhance the detection of nucleic biomarkers. Telomerase Repeat Amplification Protocol assay was used as a model for real-time PCR-based amplification and detection of DNA. Using TRAPeze XL kit, telomerase-extended DNA fragments were obtained in extracts of serial 10-fold dilutions of telomerase-positive cells, then amplified and detected during 40-cycle real-time PCR. Subsequently, characteristic 6-base DNA ladder patterns were revealed in the post-PCR samples with capillary electrophoresis (CE). In our CE instrument, fluorescently labeled DNA fragments separate in a single-capillary module and are illuminated by a fiberized Ar-ion laser. The laser-induced fluorescence (LIF) is filtered and detected by the fiberized single photon detector (SPD). To assess the sensitivity of our instrument, we performed PCR at fewer cycles (29 and 25), so that the PCR machine could detect amplification only in the most concentrated samples, and then examined samples with CE. Indeed, PCR has detected amplification in samples with minimum 10(4) cells at 29 cycles and over 10(5) cells at 25 cycles. In contrast, the SPD-based CE-LIF has revealed 6-base repeats in samples with as low as 10(2) cells after 29 cycles and 10(3) cells after 25 cycles. Thus, we have demonstrated 100- to 1000-fold increase in the sensitivity of biomarker detection over real-time PCR, making our approach especially suitable for analysis of clinical samples where abundant PCR inhibitors often cause false-negative results.

Noninvasive detection of prostate cancer by quantitative analysis of telomerase activity.

PURPOSE: Prostate cancer is the most common male malignancy and the second leading cause of male cancer death; therefore, there is urgent necessity for noninvasive assays for early detection of prostate cancer. Obtaining prostate tumor samples surgically is problematic because the malignancy is heterogeneous and multifocal and early-stage tumors are nonpalpable. In contrast, exfoliated cells represent the cancer status of the entire gland better due to the general tendency of cancer cells to exfoliate into biological fluids. The purpose of this study was to clarify whether quantitative analysis of telomerase activity in exfoliated cells in urine could serve as a reliable molecular marker of prostate malignancy. EXPERIMENTAL DESIGN: We analyzed prospectively post-prostatic examination-exfoliated cells from the urine of 56 patients undergoing routine prostate screening. Epithelial cells were isolated and enriched by immunomagnetic separation. Telomerase activity was analyzed by quantitative real-time PCR telomeric-repeat amplification protocol assay using Opticon MJ research instrument. RESULTS: We report now that all prostate cancer patients revealed high levels of telomerase activity thereby showing 100% of the assay sensitivity. In contrast, the majority of patients with clinically confirmed benign prostatic hyperplasia (BPH) did not express any telomerase activity (70% of all BPH patients), most likely presenting cancer-free cases, or expressed low levels of activity (18%). However, about 12% of BPH patients revealed high levels of telomerase activity that potentially can reflect hidden prostate cancer. CONCLUSIONS: We suggest that the quantitative analysis of telomerase activity can be useful for the selection of prostate cancer and cancer-free cases.