Higher Paclitaxel Concentrations Research Articles

Abstract CT106: First-in-human phase 1 study of paclitaxel-eluting PTM-101 film in subjects with borderline resectable or locally advanced pancreatic cancers

Abstract Background Unlike many other types of cancer, improvements in the treatment of pancreatic cancer have been very limited, in part due to the inability to deliver chemotherapy to the tumor at efficacious concentrations for a prolonged time. PTM-101 is a novel biodegradable film containing paclitaxel and is designed to continuously release high concentrations of paclitaxel precisely to the site of a pancreatic tumor over one month. Methods This first in human study was conducted at one center in Australia to assess the safety, toxicity, and surgical feasibility of a single administration of PTM-101 containing 100 mg of paclitaxel. PTM-101 was sutured directly onto the pancreatic surface overlying the tumor by a surgical oncologist using standard laparoscopic equipment during a disease-staging assessment. Approximately 3 weeks after PTM-101 placement, all participants began standard of care therapy which included mFOLFIRINOX. This study enrolled 3 subjects that had treatment naïve, borderline resectable or locally advanced, pancreatic adenocarcinoma. Subjects were monitored closely for local and systemic toxicities, as well as for preliminary signals of efficacy. An independent central imaging lab reviewed CT scans to determine changes in tumor volume. Results PTM-101 was successfully implanted over the tumor site in all three subjects. In all cases, there were no adverse events reported during the procedure. Overall, PTM-101 was well tolerated with a total of five Grade 1 adverse events judged at least possibly related to the procedure or the mFOLFIRINOX. No serious adverse events (SAEs) or deaths occurred. Paclitaxel was undetectable in the circulation at all time points. Subjects were followed for 6 months. Best overall response rate according to RECIST was stable disease (2 subjects) and partial response (1 subject). An independent analysis showed that all tumors had a reduction in size in the anterior/posterior diameter, which was consistent with unidirectional paclitaxel release from PTM-101. Two of the 3 subjects had a >30% tumor volume reduction, importantly, with the initial decrease in tumor size detected prior to mFOLFIRINOX. Conclusion PTM-101 was readily implanted during diagnostic laparoscopy and was well tolerated with no SAEs or deaths reported during 6 months of follow up. This study demonstrated that a PTM-101 implant was surgically feasible, safe, resulted in no systemic paclitaxel exposure, and caused a tumor size reduction in all 3 subjects. Additional studies may demonstrate the potential for PTM-101 to be a first-line adjunct prior to neoadjuvant therapy in treatment naïve, borderline resectable or locally advanced, pancreatic cancer. Citation Format: Charles Pilgrim, Marty Smith, Ee-Jun Ban, Samantha Ellis, John Zalcberg, Benjamin Markman, Margaret Lashof-Sullivan, Laura Indolfi. First-in-human phase 1 study of paclitaxel-eluting PTM-101 film in subjects with borderline resectable or locally advanced pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT106.

Local, Downstream, and Systemic Evaluation after Femoral Artery Angioplasty with Kanshas Drug-Coated Balloons In Vitro and in a Healthy Swine Model

PurposeTo evaluate the incidence of distal embolism and local vascular responses after treatment with the Kanshas drug-coated balloon (DCB) in a preclinical model. Materials and MethodsA total of 90 femoral arteries from 35 healthy swine were treated with a single-dose (×1) or triple-dose (×3) Kanshas DCB that applies the Unicoat technology with 3.2 μg/mm2 of paclitaxel. An uncoated Kanshas balloon was used as a control. The arterial wall, downstream skeletal muscle, and nontarget organs (kidneys, lungs, lymph nodes, liver, spleen, and heart) were histologically evaluated. For pharmacokinetic evaluation, a total of 40 healthy swine were treated with ×1 Kanshas DCB, and treated vessels were evaluated ex vivo with high-performance liquid chromatography-mass spectrometry. ResultsArteries treated with the Kanshas DCB showed mild proteoglycan deposition accompanied by the loss of smooth muscle cells (SMCs). These changes increased in a dose-dependent manner (medial SMC loss at 28 days in the ×1 vs ×3 groups, in depth, 1 (0.75–1.38) vs 2 (1.63–2.44); P = .0008; in circumference, 0.83 (0.67–1) vs 1.5 (1.19–1.81); P = .0071). No evidence of distal embolization in skeletal muscles (0 of 210 histological sections) and nontarget organs (0 of 345 sections) was observed. The pharmacokinetic evaluation showed high paclitaxel concentration in the treated artery (912 ng/mg, peaking at 3 minutes), which remained detectable at up to 180 days (0.04 ng/mg). ConclusionsThe Kanshas DCB showed a local drug effect in treated arteries up to 180 days with a high concentration of paclitaxel and no histological evidence of distal embolization.

Paclitaxel increases axonal localization and vesicular trafficking of Nav1.7.

The microtubule-stabilizing chemotherapy drug paclitaxel (PTX) causes dose-limiting chemotherapy-induced peripheral neuropathy (CIPN), which is often accompanied by pain. Among the multifaceted effects of PTX is an increased expression of sodium channel Nav1.7 in rat and human sensory neurons, enhancing their excitability. However, the mechanisms underlying this increased Nav1.7 expression have not been explored, and the effects of PTX treatment on the dynamics of trafficking and localization of Nav1.7 channels in sensory axons have not been possible to investigate to date. In this study we used a recently developed live imaging approach that allows visualization of Nav1.7 surface channels and long-distance axonal vesicular transport in sensory neurons to fill this basic knowledge gap. We demonstrate concentration and time-dependent effects of PTX on vesicular trafficking and membrane localization of Nav1.7 in real-time in sensory axons. Low concentrations of PTX increase surface channel expression and vesicular flux (number of vesicles per axon). By contrast, treatment with a higher concentration of PTX decreases vesicular flux. Interestingly, vesicular velocity is increased for both concentrations of PTX. Treatment with PTX increased levels of endogenous Nav1.7 mRNA and current density in dorsal root ganglion neurons. However, the current produced by transfection of dorsal root ganglion neurons with Halo-tag Nav1.7 was not increased after exposure to PTX. Taken together, this suggests that the increased trafficking and surface localization of Halo-Nav1.7 that we observed by live imaging in transfected dorsal root ganglion neurons after treatment with PTX might be independent of an increased pool of Nav1.7 channels. After exposure to inflammatory mediators to mimic the inflammatory condition seen during chemotherapy, both Nav1.7 surface levels and vesicular transport are increased for both low and high concentrations of PTX. Overall, our results show that PTX treatment increases levels of functional endogenous Nav1.7 channels in dorsal root ganglion neurons and enhances trafficking and surface distribution of Nav1.7 in sensory axons, with outcomes that depend on the presence of an inflammatory milieu, providing a mechanistic explanation for increased excitability of primary afferents and pain in CIPN.

Quantifying 3D cell-matrix interactions during mitosis and the effect of anticancer drugs on the interactions

The mechanical force between cells and the extracellular microenvironment is crucial to many physiological processes such as cancer metastasis and stem cell differentiation. Mitosis plays an essential role in all these processes and thus an in-depth understanding of forces during mitosis gains insight into disease diagnosis and disease treatment. Here, we develop a traction force microscope method based on monolayer fluorescent beads for measuring the weak traction force (tens of Pa) of mitotic cells in three dimensions. We quantify traction forces of human ovarian granulosa (KGN) cells exerted on the extracellular matrix throughout the entire cell cycle in three dimensions. Our measurements reveal how forces vary during the cell cycle, especially during cell division. Furthermore, we study the effect of paclitaxel (PTX) and nocodazole (NDZ) on mitotic KGN cells through the measurement of traction forces. Our results show that mitotic cells with high concentrations of PTX exert a larger force than those with high concentrations of NDZ, which proved to be caused by changes in the structure and number of microtubules. These findings reveal the key functions of microtubule in generating traction forces during cell mitosis and explain how dividing cells regulate themselves in response to anti-mitosis drugs. This work provides a powerful tool for investigating cell-matrix interactions during mitosis and may offer a potential way to new therapies for cancer.

Dual-encapsulated biodegradable 3D scaffold from liposome and waterborne polyurethane for local drug control release in breast cancer therapy

Compared with the traditional chemotherapy by injection, local release of drugs in the lesion area is a more efficient and less harmful treatment for solid tumors. However, the selection of appropriate drug carrier and controlled release of chemotherapy drugs are still great challenges. Herein, a kind of dual-encapsulated three-dimensional (3D) scaffold is designed for local drug release via blending the paclitaxel (PTX) loaded phospholipid liposomes with waterborne polyurethane (PU) by freeze-drying. The controlled release of paclitaxel is carried out through two simultaneous procedures. First, liposomes encapsulated in polyurethane scaffold can slowly release by water absorption and degradation of polyurethane. Then paclitaxel encapsulated in liposomes can also be released into water. Compared with the polyurethane scaffold which directly encapsulated paclitaxel, dual-encapsulated scaffold has slower initial release amount and maintain higher concentration of paclitaxel in later stage. Moreover, the protection of the phospholipid layer can prevent paclitaxel from being quickly decomposed and cleared, which could greatly improve the bioavailability and therapeutic effect of paclitaxel. Cell experiment results can be seen that dual-encapsulated scaffold not only has higher inhibition rate to the breast cancer MCF7 cells, but also has less damage to normal tissue cells. It provides a more effective platform for the local drug therapy in the treatment of tumors.

Antagonistic anticancer effect of paclitaxel and digoxin combination.

Despite the growing interest in the antitumor effect of cardiotonic steroids, combination treatments with well-established chemotherapy drugs like paclitaxel have been rarely investigated. Moreover, paclitaxel has been suggested as a Na+ /K+ -ATPase inhibitor. Here we investigated the effect of paclitaxel and digoxin alone or in combination on the viability of human lung (A549) and cervical cancer (HeLa) cell lines and the inhibitory effect of paclitaxel on several mammalian Na+ /K+ -ATPases. Although the viability of both tumor cell lines was concentration-dependently affected by digoxin treatment after 48 hours (A549 IC50 = 31 nM and HeLa IC50 = 151 nM), a partial effect was observed for paclitaxel, with a maximal inhibitory effect of 45% at 1000 nM with A549 and around 70% with HeLa cells (IC50 = 1 nM). Although the two drugs were cytotoxic, their combined effect in HeLa cells was revealed to be antagonistic, as estimated by the combination index. No direct inhibitory effect of paclitaxel was detected in human, pig, rat, and mouse Na+ /K+ -ATPase enzymes, but high concentrations of paclitaxel decreased the Na+ /K+ -ATPase activity in HeLa cells after 48 hours without affecting protein expression. Our findings demonstrate that, under our conditions, paclitaxel and digoxin cotreatment produce antagonistic cytotoxic effects in HeLa cells, and the mechanism of action of paclitaxel does not involve a direct inhibition of Na+ /K+ -ATPase. More studies shall be designed to evaluate the consequences of the interaction of cardiotonic steroids and chemotherapy drugs.

Rate of Drug Coating Dissolution Determines In-Tissue Drug Retention and Durability of Biological Efficacy.

Two different drug-coated balloons (DCBs) possessing different coating formulations were compared for rate of coating dissolution in vitro, in addition to tissue drug concentration and histological responses of treated vascular tissue in vivo, to determine if the rate of drug bioavailability to vascular tissue can impact the degree and duration of the observed pharmacological response to locally delivered drug. In vitro dissolution comparison demonstrated that a urea/paclitaxel-based coating formulation (IN.PACT™ Admiral™) released drug from solid to soluble phase at a slower and constant rate, yielding approximately 7% solubilized drug in 24 h. In contrast, a coating formulated from polysorbate/sorbitol/paclitaxel (Lutonix™) released 51% of solid phase drug to soluble phase in 1 h of dissolution with the remainder solubilizing in 24 h. In vivo evaluation of tissue drug concentration of both products showed significantly different tissue pharmacokinetic profile, with a higher concentration of paclitaxel in tissue at 90 days with a urea-based formulation excipient. Histological comparison of smooth muscle cell loss in response to drug exposure revealed contrasting trends of smooth muscle cell loss from 28 to 90 days with significantly higher response to drug observed at 90 days with the urea-based formulation. Rapid dissolution of drug from the polysorbate/sorbitol coating formulation was associated with an early increase in local cellular response to drug which diminished over 90 days with clearance of local drug from tissue. Sustained long-term drug-in-tissue concentration associated with the urea-based formulation demonstrated sustained pharmacological activity at 90 days, suggesting that slow coating dissolution provides a sustainable long-term tissue response.

Abstract A58: Transcription factor SREBP2 mediates ovarian cancer drug resistance and recurrence

Abstract Ovarian cancer, the most lethal gynecologic malignancy, has a poor prognosis. Existing chemotherapies are challenged by the emergence of drug resistance and metastasis of tumor cells, which obstruct sustained, relapse-free patient cures. Resistance can emerge from pre-existing mutations, secondary mutations, or other nongenetic mechanisms or metabolic adaptations. SREBPs are necessary for cell survival in low-nutrient conditions. SREBP1 predominantly regulates fatty acid and glycolysis while SREBP2 regulates cholesterol metabolism. SREBP2 is a sterol sensitive transcription factor regulating cholesterol homeostasis including the expression of LDLR and HMGCR. Recent reports suggest that SREBP2 may promote mesenchymal and stem-cell states. These observations support our working hypothesis that the regulation of cholesterol and fatty acids through SREBP2 is critical for drug tolerance, resistance, and viability of a cancer stem cell population. We further hypothesize that drug-tolerant ovarian cancer cells depend on SREBP2 to re-enter the cell cycle and proliferate. To test this hypothesis, we assessed the importance of SREBP2 in an in vitro drug tolerant model. Ovarian cancer cells were treated with high concentrations of paclitaxel for 1 week and the surviving cells were examined. SREBP2, HMGCR, and related factors were significantly upregulated in these surviving drug-tolerant cells. To evaluate the function of SREBP2 in drug-tolerant cells, stable cell lines targeting the SREBF2 gene using CRISPR technology were created in OVCAR8. Using this SREBF2-KD line, we assessed the response of ovarian cancer cells to drug treatment under different environments. SREBF2-KD cells were more sensitive to statin and paclitaxel in low-sterol environments compared to control cells. These observations highlight the need for SREBP2 in stressed conditions for cancer cells to survive drug treatment. Moreover, after treatment with high concentrations of paclitaxel, SREBF2-KD cells grew back significantly slower compared to control cells. To characterize the mechanisms controlled by SREBP2, we examined the effects on the cell cycle. We have observed a significant change in the distribution of the cell cycle phases between SREBF2-KD and control cells treated with paclitaxel, which suggests an important role for SREBP2 in ovarian cancer cells’ exit from cell cycle arrest after chemotherapy. To further examine the role of SREBP2 in drug resistance and tumor relapse, we are currently extending these in vitro observations to in vivo models. In summary, our study illustrates the crucial role of cholesterol pathway for ovarian cancer not only under stress conditions such as chemotherapy, but also in return to a high proliferative state upon recurrence. These data suggest that targeting SREBP2 may represent a new approach for sequential therapy to eliminate cancer cells that survive initial treatment. Citation Format: Galina Karashchuk, Alexander S. Brodsky. Transcription factor SREBP2 mediates ovarian cancer drug resistance and recurrence. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A58.

Abstract 4409: Cholesterol pathway determines ovarian cancer drug resistance through transcription factor SREBP2

Abstract Ovarian cancer is the most common cause of gynecological cancer death in women in United States. Up to 70% of all ovarian cancer cases are high-grade serous carcinomas with 5-year survival rates less than 30%. Recent studies have suggested the importance of the cholesterol pathway in multiple cancers including gynecological malignancies. Here we demonstrate that genetic or pharmacological disturbance of cholesterol pathway in high-grade serous ovarian carcinoma cell lines results in significant changes in survival rate after drug treatment, and in protein and RNA expression patterns. SREBP2 is a transcription factor encoded by SREBF2 gene that regulates expression of sterol-regulated genes and thus maintains cholesterol homeostasis. To assess the role of SREBP2 in ovarian cancer we have created a stable OVCAR8 cell line with SREBF2 disrupted using CRISPR technology. This SREBF2-KD line has reduced SREBF2 mRNA and protein expression level that indicates an effective gene knockdown. Activation of SREBF2 is dependent on the cholesterol status of the cell. We observed that expression of the SREBP2 precursor form is significantly reduced in SREBF2-KD line when cells are maintained under low serum conditions. Mutant cells treated with paclitaxel in low, but not high serum or in presence of statin, revealed significantly lower cell viability. SREBF2-KD cells do not survive long-term, high concentrations of paclitaxel, nor do they grow back as efficiently as control cells. RNA expression and proteomic analysis revealed the critical regulators mediating SREBP2 activity in stressed cells. Together, these observations suggest that the cholesterol pathway is critical for ovarian cancer cells not only to resist stresses, such as chemotherapy, but also to return to a high proliferation state upon recurrence. Further studies will provide important targets for developing new drugs for treatment of ovarian cancer. Citation Format: Galina Karashchuk, Nataliya Karashchuk, Signe Caksa, Tyler S. Smith, Alexander S. Brodsky. Cholesterol pathway determines ovarian cancer drug resistance through transcription factor SREBP2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4409. doi:10.1158/1538-7445.AM2017-4409

Paclitaxel nanoparticle awakens immune system to fight against cancer

A high concentration of paclitaxel (PTX) is used as an anti-tumor chemotherapy but is toxic to immune cells. At lower concentrations, PTX was found able to stimulate the anti-tumor potentials of immune cells. Thus, decreasing the cytotoxicity of PTX at high concentration while maintaining its anti-tumor stimulation to immune cells remains challenging. Herein, by employing a click condensation reaction, we rationally designed a PTX derivative, Cys(StBu)-Arg-Arg-Arg-Lys(PTX)-CBT (1), for the facile preparation of its nanoparticle 1-NP. In vitro assays indicated that, at high PTX concentrations, 1-NP showed significantly lower cytotoxicity to macrophages than did PTX, and could be efficiently phagocytosed by macrophages and consequently polarize the cells into an anti-tumor state in a dose-dependent manner. In vivo experiments further confirmed that 1-NP had a higher anti-tumor efficacy than did free PTX but lower cytotoxicity to immune cells in both immune organs and tumor sites. Our results suggest that, by using different doses of 1-NP, patients can precisely regulate the activation of the immune system for an effective anti-tumor and balanced autoimmune responses. We also envision that our strategy could lead to a combined use of immunotherapy and chemotherapy for a more efficient anti-tumor treatment in the future.

Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1.

The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.

Vitrification of mouse MII oocytes: Developmental competency using paclitaxel

ObjectiveOocyte cryopreservation provides an important alternative for fertility preservation for women who will be treated with cytotoxic drugs. However, it can cause spindle disorganization of microtubules, putting the zygote at risk for aneuploidy. Paclitaxel is known to stabilize the microtubules that constitute the spindle. The aim of this study was to investigate the suitable concentration of paclitaxel for adding to the vitrification media to improve the developmental potential of post-thawed mature oocytes to blastocyst formation in mice. Materials and MethodsA total of 300 MII oocytes were retrieved from superovulated mice, and were divided into three groups of control, Experimental I, and Experimental II. Oocytes in Experimental I and Experimental II were cryopreserved in the presence of 0.5μM or 1μM of paclitaxel in vitrification media, respectively. After thawing, all oocytes were incubated in G-IVF medium for 1 hour. From each group,12 oocytes were selected for viability evaluation by Hoechst/propidium iodide nuclear staining. Standard in vitro fertilization was performed on the rest of the oocytes and embryo development was followed to the blastocyst stage. ResultsFertilization rate was not significantly different between the three groups. However, the cleavage rate (55%) in Experimental II group was significantly lower compared to Experimental I (88%) and control groups (83%). There was a detectable difference between the three groups at the blastocyst rate (Experimental I and control groups, p = 0.004; Experimental II vs. control and Experimental I, p < 0.001). The highest rates of parthenogenesis and arrest were in Experimental II (16% and 21%, respectively) compared with control (6% and 5%, respectively) and Experimental I (5% and 3%, respectively). There was also a significant decrease in viability rate of oocytes in Experimental II compared to the other groups. ConclusionA high concentration of paclitaxel, an anticancer drug, interrupted the mouse oocyte competency when supplemented to vitrification media. Consequently, the optimal concentration of this cytoskeleton stabilizer may improve the post-thawed developmental abilities of oocytes.

Amphiphilic Triblock Copolymers from Poly(2‐oxazoline) with Different Hydrophobic Blocks: Changes of the Micellar Structures upon Addition of a Strongly Hydrophobic Cancer Drug

Amphiphilic triblock copolymers with poly(2‐methyl‐2‐oxazoline) (MeOx) end blocks and a poly(2‐n‐butyl‐2‐oxazoline) (nBuOx) middle block have been found to solubilize large amounts of the cancer drug paclitaxel (PTX) whereas this is not the case when the middle block is more hydrophobic, such as poly(2‐n‐nonyl‐2‐oxazoline) (NOx) (Schulz et al., ACS Nano2014, 8, 2686). To further elucidate the origin of this behavior, dilute aqueous solutions (5–10 mg mL−1) of these two copolymers have been investigated by small‐angle neutron scattering (SANS), both in the absence and presence of PTX. Whereas PMeOx‐b‐PNOx‐b‐PMeOx forms mainly worm‐like micelles coexisting with large aggregates, spherical micelles are predominant in PMeOx‐b‐PnBuOx‐b‐PMeOx. Already small PTX concentrations lead to shape changes in PMeOx‐b‐PNOx‐b‐PMeOx toward spherical micelles. In contrast, changes in the aggregation behavior of PMeOx‐b‐PnBuOx‐b‐PMeOx are only observed at higher PTX concentrations with a transformation into raspberry‐like particles, which may explain the high PTX loading capacity of PMeOx‐b‐PnBuOx‐b‐PMeOx micelles. image

Abstract 4453: The dual CK2/TNIK inhibitor, ON108600 targets cancer stem cells and induces apoptosis of paclitaxel resistant triple-negative breast cancer cells

Abstract Triple negative breast cancer (TNBC) is associated with a poor prognosis and high frequency of recurrence. Because the molecular mechanisms that are deregulated in this tumor type are not well understood, there is a lack of targeted therapies that can effectively treat this disease. An unfortunate limitation of existing TNBC therapies is the frequency of relapse, which is highly resistant and metastatic and has been attributed to tumor-initiating stem cells (T-ICs). In patients with relapsed TNBC, T-ICs with a CD44high/CD24-/low antigenic phenotype are enriched in the tumor cell population. Our previous data illustrated that a small molecule kinase inhibitor, ON108600, potently inhibited the survival and growth of TNBC cell lines and mouse xenografts. To investigate whether ON108600 has a similar inhibitory effect on T-ICs, we performed clonogenic survival assays using sorted CD44high CD24-/low cells isolated from TNBC cell lines. ON108600 potently inhibited the stem cell activity and self-renewal ability of these T-ICs. Although paclitaxel (PTX) treatment improves survival of TNBC patients, acquired resistance to this drug is a common occurrence. Furthermore, strategies that target PTX resistant cells remain elusive. To investigate the molecular mechanisms underlying acquired PTX-resistance in TNBC and evaluate the efficacy of ON108600, we generated PTX-resistant cell lines using the MDA-MB-231 and BT-20 TNBC cell lines. Drug-resistant cells were established by exposure to increasing concentrations of Paclitaxel, and resistance was validated by cell viability and colony formation in the presence of high concentrations of PTX. PTX-resistant MDA-MB-231 and BT-20 cells exhibited an approximate 1000-fold increase in resistance to PTX as compared to the parental cells. Importantly, PTX-resistant TNBC cells displayed a stem-like phenotype characterized by loss of epithelial differentiation markers (e-Cadherin, CD24) and a gain of epithelial-mesenchymal transition markers (N-Cadherin, CD44, Oct4, Snail). Kinase profiling studies have indicated that ON108600 targets kinases CK2, and Traf2- and Nck-interacting kinase, TNIK. Although CK2 subunit levels in resistant cells were unchanged, PTX-resistant cells showed a marked upregulation of TNIK, an activating kinase for T-cell factor-4 (TCF-4) and consequently, a marked increase in β-catenin and Wnt target genes Axin-2 and Cyclin D1. We therefore examined whether dual inhibition of CK2 and TNIK was effective in killing PTX-resistant cells. Treatment of PTX-resistant TNBC cells with ON108600 resulted in marked increase in apoptosis and inhibition of clonogenic survival and mamosphere forming ability of these cells, suggesting that dual CK2/TNIK inhibition may be an effective way to overcome PTX-resistance in TNBC. We are currently testing the efficacy of ON108600 in paclitaxel resistant xenograft models. Citation Format: Amol Padgaonkar, Stephen Cosenza, Venkat Pallela, Venkata Subbaiah DRC, MV Ramana Reddy, E Premkumar Reddy. The dual CK2/TNIK inhibitor, ON108600 targets cancer stem cells and induces apoptosis of paclitaxel resistant triple-negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4453. doi:10.1158/1538-7445.AM2015-4453

Improving paclitaxel delivery: in vitro and in vivo characterization of PEGylated polyphosphoester-based nanocarriers.

Nanomaterials have great potential to offer effective treatment against devastating diseases by providing sustained release of high concentrations of therapeutic agents locally, especially when the route of administration allows for direct access to the diseased tissues. Biodegradable polyphosphoester-based polymeric micelles and shell cross-linked knedel-like nanoparticles (SCKs) have been designed from amphiphilic block-graft terpolymers, PEBP-b-PBYP-g-PEG, which effectively incorporate high concentrations of paclitaxel (PTX). Well-dispersed nanoparticles physically loaded with PTX were prepared, exhibiting desirable physiochemical characteristics. Encapsulation of 10 wt% PTX, into either micelles or SCKs, allowed for aqueous suspension of PTX at concentrations up to 4.8 mg/mL, as compared to <2.0 μg/mL for the aqueous solubility of the drug alone. Drug release studies indicated that PTX released from these nanostructures was defined through a structure-function relationship, whereby the half-life of sustained PTX release was doubled through cross-linking of the micellar structure to form SCKs. In vitro, physically loaded micellar and SCK nanotherapeutics demonstrated IC50 values against osteosarcoma cell lines, known to metastasize to the lungs (CCH-OS-O and SJSA), similar to the pharmaceutical Taxol formulation. Evaluation of these materials in vivo has provided an understanding of the effects of nanoparticle structure-function relationships on intratracheal delivery and related biodistribution and pharmacokinetics. Overall, we have demonstrated the potential of these novel nanotherapeutics toward future sustained release treatments via administration directly to the sites of lung metastases of osteosarcoma.

Establishment and characterization of a paclitaxel-resistant human esophageal carcinoma cell line

The aim of this study was to establish a new paclitaxel (PTX)-resistant human esophageal squamous carcinoma (ESCC) cell line and investigate its biological characteristics. The resistant cell line (EC109/Taxol) was developed in vitro by intermittent exposure of the human ESCC cell line EC109 to a high concentration of PTX with time-stepwise increment over a period of 6 months. The MTT assay was performed to test the drug resistance of EC109 and EC109/Taxol cells. The morphological features were observed using inverted microscopy and apoptosis was measured by flow cytometry (FCM) and Hoechst 33258 fluorescence staining. Cell growth curves and colony formation of EC109 and EC109/Taxol cells were compared. FCM was also used to determine the distribution of the cell cycle. The protein levels of Bcl-2, Bax, Procaspase-3 and P-gp were detected by western blotting. P-gp activity was evaluated by Rh123 accumulation and efflux assay. In vivo resistance characterization was investigated. EC109/Taxol cells were 67.2-fold resistant to PTX in comparison with EC109 cells, and also exhibited cross-resistance to 5-fluorouracil (5-FU), cisplatin (CDDP) and epirubicin (EPI). FCM and Hoechst 33258 fluorescence staining confirmed that EC109 cells treated with PTX showed significantly higher percentage of apoptotic cells compared to EC109/Taxol cells. Simultaneously, EC109/Taxol cells exhibited changes in morphology, proliferation rate, doubling time, cell cycle distribution and colony formation rate were detected as compared with EC109 cells. The resistant cell line overexpressed Bcl-2, Procaspase-3 and P-gp protein, and showed decreased Bax expression. Further, EC109/Taxol cells did not change PTX resistance in vivo. This is the first report on the establishment of an EC109/Taxol cell line with higher resistance. Bcl-2, Bax, Procaspase-3 and P-gp are involved in the resistance of cell lines to PTX, which are invaluable tools to study the resistance of anticancer drugs and to identify the methods to overcome resistance.

Modeling Canada yew (Taxus canadensis Marsh.) distribution and abundance in the boreal forest of northeastern Ontario, Canada

Canada yew (Taxus canadensis Marsh.) is a gymnosperm shrub that occurs in the understorey of coniferous and deciduous forests throughout the northeastern United States and southeastern Canada. Canada yew foliage contains high concentrations of paclitaxel and other taxanes, which have proven effective in treating many forms of cancer. A major challenge to the sustainable management of wild Canada yew harvest is the lack of a spatial inventory of the resource. The objective of this study was to develop a spatial model to predict the distribution and abundance of Canada yew populations in a forest landscape representative of the boreal forest region of Ontario, Canada. The study was conducted in the Romeo Mallete Forest (RMF) located on the southern edge of the boreal forest west of Timmins, Ontario. Abundance of Canada yew populations was assessed in 140 circular sample plots (5541m2), established at locations throughout the RMF. Canada yew abundance in each sample plot was estimated as the mean percent cover of three randomly located 100m2 subplots. Predictor variables were extracted from the Forest Resources Inventory (FRI) of the RMF, and from layers derived from remote sensing data covering the study area. A combination of classification and regression trees with Random Forests analysis was used to develop a classification model for FRI polygons based on patterns of Canada yew abundance, and assess the performance of the model. The classification error rate on the presence/absence model was approximately 21%, and the root mean squared error on the best continuous cover model was 9.1%. In all analyses, the most important classification node was based on percent basal area of the shade-tolerant late successional species Balsam Fir (Abies balsamea (L.) Mill), which suggests that patterns of Canada yew abundance are driven largely by time since disturbance.

A new bioreactor for paclitaxel production based on foam separation

ABSTRACTWe report here the development of a new bioreactor (bioreactor with foam separation, BFS) for efficient production and recovery of paclitaxel that circumvents paclitaxel's inhibitory effects on growth through a feedback mechanism. The BFS captures paclitaxel through its hydrophobic interaction with the foam generated by aerated culture medium. The BFS comprises co‐axial 0.65 dm3 outer (150 mm height and 75 mm diameter) and 0.15 dm3 inner vessels (90 mm height and 50 mm diameter) equipped with a ball‐type sparger (5–10 µm pore size, 10 mm diameter) and a wire‐framed draft tube covered by a water‐permeable cellulose bag (150 × 40 mm, 100 µm maximum pore size). The BFS was tested by adding paclitaxel alone to the medium of 0.12 dm3 in the inner vessel and collecting it in the foam that overflowed into the outer vessel. Paclitaxel was separated with first‐order kinetics and was optimally recovered using an aeration rate of 0.05 dm3 min−1 (0.21 vvm). Next, a Taxus cuspidata callus was cultured using the optimal aeration rate. A conventional shaking culture (110 rpm) served as the control using a 0.2 dm3 Erlenmeyer flask (0.12 dm3 medium). Paclitaxel was maintained at less than 0.02 g m−3 in the inner vessel of the BFS. Consequently, there was no feedback inhibition, and the paclitaxel production increased by a factor of five in the BFS compared with the control culture where there was feedback inhibition by the high concentration of paclitaxel. © 2013 Curtin University of Technology and John Wiley &amp; Sons, Ltd.

Paclitaxel and CYC3, an aurora kinase A inhibitor, synergise in pancreatic cancer cells but not bone marrow precursor cells

Background:Amplification of aurora kinase A (AK-A) overrides the mitotic spindle assembly checkpoint, inducing resistance to taxanes. RNA interference targeting AK-A in human pancreatic cancer cell lines enhanced taxane chemosensitivity. In this study, a novel AK-A inhibitor, CYC3, was investigated in pancreatic cancer cell lines, in combination with paclitaxel.Methods:Western blot, flow cytometry and immunostaining were used to investigate the specificity of CYC3. Sulforhodamine B staining, time-lapse microscopy and colony-formation assays were employed to evaluate the cytotoxic effect of CYC3 and paclitaxel. Human colony-forming unit of granulocyte and macrophage (CFU-GM) cells were used to compare the effect in tumour and normal tissue.Results:CYC3 was shown to be a specific AK-A inhibitor. Three nanomolar paclitaxel (growth inhibition 50% (GI50) 3 nℳ in PANC-1, 5.1 nℳ in MIA PaCa-2) in combination with 1 μℳ CYC3 (GI50 1.1 μℳ in MIA PaCa2 and 2 μℳ in PANC-1) was synergistic in inhibiting pancreatic cell growth and causing mitotic arrest, achieving similar effects to 10-fold higher concentrations of paclitaxel (30 nℳ). In CFU-GM cells, the effect of the combination was simply additive, displaying significantly less myelotoxicity compared with high concentrations of paclitaxel (30 nℳ; 60–70% vs 100% inhibition).Conclusion:The combination of lower doses of paclitaxel and CYC3 merits further investigation with the potential for an improved therapeutic index in vivo.

Abstract 1924: The aurora kinase inhibitor CYC3 synergizes with low concentrations of paclitaxel in pancreatic cancer cells in vitro

Abstract CYC3, an Aurora Kinase A specific inhibitor, suppresses the pancreatic cancer cell growth (72h GI50 2.4μM in MiaPaCa-2 cells and 1.95μM in Panc-1 cells), arrest the cells at M phase and induces apoptosis. In order to evaluate CYC3's clinical potential, we employed mathematical models and simulations to look for possible synergistic combinations of CYC3 with paclitaxel in MiaPaCa-2 and Panc-1 cells. We have identified low concentrations of paclitaxel (3nM) and CYC3 is synergistic in inhibiting pancreatic cell growth, achieving similar effects as high concentrations of paclitaxel (30nM). Liquid Chromatography-Mass Spectrometry (LC-MS) analysis shows that CYC3 does not alter the cellular uptake of paclitaxel, supporting a synergistic mechanism at molecular level. In addition, the combination of CYC3 with low concentration of paclitaxel (3nM) displays less myelotoxicity compared to high concentrations of paclitaxel (30nM) in colony formation assay using Colony Forming Unit of Granulocyte and Macrophage (CFU-GM) (60-70% vs 100% inhibition), suggesting a potentially safer but equally efficient application of paclitaxel in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1924. doi:1538-7445.AM2012-1924