Antitumor Effect Of Paclitaxel Research Articles

Abstract 1556: CDKN3 inhibition enhances the antitumor effect of paclitaxel by promoting abnormal mitosis of cancer cells

Abstract Background: Paclitaxel (PTX) is one of the key drugs in clinical practice for esophageal squamous cell carcinoma (ESCC), however, therapeutic effects of PTX alone are not sufficient. PTX causes activation of spindle assembly checkpoint resulting in mitotic arrest of cancer cells. Recently, the antitumor effect of PTX has been shown to depend on aberrant chromosome segregation and abnormal spindle formation. Whereas, recent reports showed that cyclin dependent kinase inhibitor 3 (CDKN3) was often overexpressed in various human cancer tissues which was attributed to the higher mitotic activity in cancer cells, and loss of CDKN3 induced abnormal mitosis and centrosome overduplication. Therefore, we hypothesized that the combination of PTX and CDKN3 inhibition may promote mitotic errors in cancer cells. The aim of this study is to examine the combined effect of PTX and CDKN3 inhibition on ESCC. Methods: The expression levels of CDKN3 were compared between ESCC cell lines (TE1, TE5, TE6, TE8, TE10, and TE11) and normal esophageal epithelial cell line (EPC2-hTERT). CDKN3 knockdown ESCC cells using shRNA were created. Cell viability assay (WST-1 and clonogenic assay), immunofluorescence microscopy for measuring the number of centrosomes or spindle microtubules, and time-lapse images for assessing mitosis and cell fate were performed. Results: The expression levels of CDKN3 in ESCC cells were high compared to normal esophageal epithelial cells. Here, TE6 and TE10 ESCC cells were selected for further experiments based on higher CDKN3 expression. WST-1 assays showed an increased sensitivity to PTX after CDKN3 knockdown. Clonogenic assay showed reduced clonogenicity with CDKN3 knockdown, which was further reduced when exposed to 1 nM PTX (no interaction by two-way ANOVA analysis). Quantification of centrosomes revealed an increased frequency of centrosome overduplication in cells with CDKN3 knockdown, and it was further increased when treated with 1 nM PTX. Quantification of microtubules revealed CDKN3 knockdown cells with 1nM PTX had irregular multipolar spindle formation in almost half of the cells in mitosis. In time-lapse images, mitotic errors were more frequent ( > 80% in combination vs 54% in PTX vs 32% in CDKN3 knockdown) and most of the daughter cells after abnormal mitoses resulted in cell death in interphase or did not undergo another cell division. The cumulative cell death was significantly increased in combination treatment with PTX and CDKN3 inhibition compared to PTX treatment alone (Hazard ratio 2.22; 95% CI 1.43 - 3.45, p = 0.0004 ) and CDKN3 inhibition alone (Hazard ratio 3.09; 95% CI 1.92 - 4.98, p < 0.0001). Conclusions: CDKN3 knockdown increased the frequencies of centrosome overduplication and multipolar spindle formation when combined with PTX exposure. These mitotic errors caused multipolar divisions and subsequent cell death. Targeting CDKN3 could be a novel anti-cancer strategy. Citation Format: Shigeki Kataoka, Junichi Matsubara, Trang H. Vu, Tomohiro Kondo, Tomoki Saito, Yosuke Mitani, Osamu Kikuchi, Atsushi Yamada, Shinya Ohashi, Manabu Muto. CDKN3 inhibition enhances the antitumor effect of paclitaxel by promoting abnormal mitosis of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1556.

Paclitaxel antitumor effect improvement in lung cancer and prevention of the painful neuropathy using large pegylated cationic liposomes

Paclitaxel (PTX), a drug widely used in lung cancer, has serious limitations including the development of peripheral neurotoxicity, which may lead to treatment discontinuation and therapy failure. The transport of PTX in large cationic liposomes could avoid this undesirable effect, improving the patient’s prognosis. PTX was encapsulated in cationic liposomes with two different sizes, MLV (180−200 nm) and SUV (80−100 nm). In both cases, excellent biocompatibility and improved internalization and antitumor effect of PTX were observed in human and mice lung cancer cells in culture, multicellular spheroids and cancer stem cells (CSCs). In addition, both MLV and SUV with a polyethylene glycol (PEG) shell, induced a greater tumor volume reduction than PTX (56.4 % and 57.1 % vs. 36.7 %, respectively) in mice. Interestingly, MLV-PEG-PTX did not induce either mechanical or heat hypersensitivity whereas SUV-PEG-PTX produced a similar response to free PTX. Analysis of PTX distribution showed a very low concentration of the drug in the dorsal root ganglia (DRG) with MLV-PEG-PTX, but not with SUV-PEG-PTX or free PTX. These results support the hypothesis that PTX induces peripheral neuropathy by penetrating the endothelial fenestrations of the DRG (80−100 nm, measured in mice). In conclusion, our larger liposomes (MLV-PEG-PTX) not only showed biocompatibility, antitumor activity against CSCs, and in vitro and in vivo antitumor effect that improved PTX free activity, but also protected from PTX-induced painful peripheral neuropathy. These advantages could be used as a new strategy of lung cancer chemotherapy to increase the PTX activity and reduce its side effects.

Abstract 3579: Pimozide combined with paclitaxel demonstrates a significant antitumor effect in head and neck squamous cell carcinoma

Abstract Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the world. Despite the development of various treatments, the five-year survival rate for HNSCC is about 50-60% and has not been improved in recent years. Immune checkpoint inhibitors are promising, but the effect of those is limited. The development of new reagents having different mechanisms against tumor progression is still needed. From the result of drug screening, Pimozide (PMZ), an antipsychotic drug used for the treatment of schizophrenia and chronic psychosis, demonstrated a strong antitumor ability to HNSCC cells expressing high level of CD44v. In this study, we examined the combined effects of PMZ and paclitaxel (PTX) for HNSCC cells. Methods: Using HNSCC cell lines (FaDu, Detroit562, HSC-2, OSC-19), the antitumor effects of PTX, PMZ, and the combination of PTX and PMZ were evaluated by MTS assay. Moreover, the reactive oxygen species (ROS) expression level under each drug administration was measured by flow cytometry. Moreover, PTX, PMZ (0.45mg/kg), PMZ (1mg/kg), PTX+PMZ were administered to the xenograft model by OSC-19. The tumor was resected after those treatments. The expression of CD44v, ALDH3A1, and NRF2 was evaluated by immunohistochemistry (IHC) of FFPE tissues from those extracted tumors. Results: PMZ single-agent inhibited tumor growth in various tumor cell lines, and the antitumor effect was enhanced by concomitant use of PTX. PMZ, which significantly increased ROS of tumor cells, was more effective than PTX in OSC-19, which was a chemoresistant cell line expressing high CD44v. Xenograft model exhibited that tumor growth was suppressed in PTX+PMZ group than in PTX and PMZ single-agent group. IHC staining revealed that PMZ administration restrained CD44v expression, and ALDH3A1 expression markedly decreased in PMZ treated tumors, but not in PTX treated tumors. Conclusions: PMZ dramatically induced ROS production in tumor cells. These data suggest that aldehyde produced by ROS might suppress HNSCC tumor growth. The results from this study indicated that PMZ could be a novel therapeutic reagent for HNSCC in combination with existing anticancer drugs. Citation Format: Shintaro Nakamura, Hiroyuki Ozawa, Juntaro Yamasaki, Shogo Okazaki, Momoko Yoshikawa, Tomoya Soma, Seiji Asoda, Osamu Nagano, Mariko Sekimizu, Shin Saito, Keisuke Yoshihama, Takuya Mikoshiba, Kaoru Ogawa, Hideyuki Saya. Pimozide combined with paclitaxel demonstrates a significant antitumor effect in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3579.

PI3K-targeting strategy using alpelisib to enhance the antitumor effect of paclitaxel in human gastric cancer

PIK3CA mutations are frequently observed in various human cancers including gastric cancer (GC). This study was conducted to investigate the anti-tumor effects of alpelisib, a PI3K p110α-specific inhibitor, using preclinical models of GC. In addition, the combined effects of alpelisib and paclitaxel on GC were evaluated. Among the SNU1, SNU16, SNU484, SNU601, SNU638, SNU668, AGS, and MKN1 GC cells, three PIK3CA-mutant cells were predominantly sensitive to alpelisib. Alpelisib monotherapy decreased AKT and S6K1 phosphorylation and induced G0/G1 phase arrest regardless of PIK3CA mutational status. The alpelisib and paclitaxel combination demonstrated synergistic anti-proliferative effects, preferentially on PIK3CA-mutant cells, resulting in increased DNA damage response and apoptosis. In addition, alpelisib and paclitaxel combination potentiated anti-migratory activity in PIK3CA-mutant cells. Alpelisib partially reversed epithelial–mesenchymal transition markers in PIK3CA-mutant cells. In a xenograft model of MKN1 cells, the alpelisib and paclitaxel combination significantly enhanced anti-tumor activity by decreasing Ki-67 expression and increasing apoptosis. Moreover, this combination tended to prolong the survival of tumor-bearing mice. Our data suggest promising anti-tumor efficacy of alpelisib alone or in combination with paclitaxel in PIK3CA-mutant GC cells.

Paclitaxel inhibits proliferation and promotes apoptosis through regulation ROS and endoplasmic reticulum stress in osteosarcoma cell

Paclitaxel is a chemotherapeutic drug for cancer, which is isolated from the Pacific yew tree. However, and the molecular mechanism and the antitumor effects of paclitaxel on osteosarcoma cell remain to be explored. The aim of our study was to explore the possible molecular mechanisms of apoptosis in osteosarcoma induced by paclitaxel. Paclitaxel can obviously decrease the proliferation of HOS-732 cells in a dose-dependent manner. Paclitaxel could induce the cell cycle arrest at the G2/M-phase and decreases the CDK5 and CCNE1 expression in HOS-732 cells. Paclitaxel promotes cell apoptosis in HOS-732 cells, which may be contacted to the decreasing of Bcl-2 protein expression. Further, the production of ROS in HOS-732 cells was remarkably increased with the increasing concentration of paclitaxel. Moreover, paclitaxel induces the ER-stress related gene and protein expression (GRP79, DDIT3 mRNA and GRP78, XBP-1 s, IRE1α protein expression) in osteosarcoma cells. Paclitaxel can inhibit the proliferation of HOS-732 cells and increase ROS and ER-stress response to promote cell apoptosis, suggesting that paclitaxel may represent a new therapeutic option for the treatment and prevention osteosarcoma.

Abstract 3818: BGJ398, a pan-FGFR inhibitor, overcomes paclitaxel resistance in urothelial carcinoma with FGFR1 overexpression

Abstract Introduction: Paclitaxel (PTX) is commonly used to treat urothelial carcinoma (UC) after platinum-based chemotherapy has failed. However, single-agent taxane therapy is not sufficient to inhibit tumor progression and drug resistance in advanced UC. Epithelial-to-mesenchymal transition (EMT) induced by fibroblast growth factor receptor (FGFR)1 signaling has been proposed as a mechanism of PTX resistance, but it is unclear whether this can be overcome by FGFR1 inhibition. The present study investigated whether FGFR1 overexpression contributes to PTX resistance and whether FGFR inhibition can enhance PTX efficacy in UC. Materials and Methods: UC cell lines: UMUC-14, RT4, T24, J82, HTB5 and HTB9 cells were used. Cells were treated with various concentration of PTX (0 to 20 nM) and BGJ398 (0 to 6 μM). The half-maximal inhibitory concentrations (IC50) were measured by the CellTiter-Glo assay. Cell cycle was analyzed after propidium iodide (PI) staining via flow cytometry. Changes in cell cycle, apoptosis and EMT-associated protein levels were assessed by Western Blot analysis. The migratory and colony formative property were analyzed by wound healing assay and colony forming assay, respectively. RNA interference was performed to assess the dependence of PTX sensitivity on pro-apoptotic proteins. Results: We showed that mesenchymal-type T24 and J82 cell lines were highly resistant to PTX (0 to 20 nM) or BGJ398 (0 to 6 μM) monotherapy compared to FGFR3-positive epithelial-type (i.e., RT4 and UMUC-14) UC cell lines. We further found that PTX (10 nM) combined with BGJ398 (6 μM) caused synergistic effects on sub G1 accumulation and apoptosis compared to PTX or BGJ398 monotherapy in T24 and J82 cells, which were accompanied by downregulation of cyclin D1 protein and upregulation of gamma-histone 2A family member X, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP). Additionally, 10 nM of PTX combined with 6 μM of BGJ398 synergistically suppressed mesenchymal-type UC cell migration and colony formation via downregulation of EMT-associated factors including snail, slug and ZEB1. FGFR1 knockdown enhanced the antitumor effect of PTX (10 nM) by upregulation of apoptosis-associated markers, cleaved caspase-9 and PARP. Conclusions: We investigated the mechanism of resistance to PTX in UC mediated by FGFR1 and found that combined treatment with BGJ398 can enhance the efficacy of PTX in UC cell lines with mesenchymal features. Our results demonstrate that combination with FGFR1-targeted therapy improves the antitumor efficacy of standard cytotoxic chemotherapy, which is a strategy that warrants further investigation in clinical trials in selected UC patients with FGFR1 overexpression. Citation Format: Haram Ryu, Se Hyun Kim, Chan-Young Ock, Koung Jin Suh, Ji Yun Lee, Ji-Won Kim, Jin Won Kim, Jeong-Ok Lee, Yu Jung Kim, Keun-Wook Lee, Soo-Mee Bang, Jee Hyun Kim, Jong Seok Lee, Joong Bae Ahn, Kui-Jin Kim, Sun Young Rha. BGJ398, a pan-FGFR inhibitor, overcomes paclitaxel resistance in urothelial carcinoma with FGFR1 overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3818.

Paclitaxel Reduces Tumor Growth by Reprogramming Tumor-Associated Macrophages to an M1 Profile in a TLR4-Dependent Manner.

Paclitaxel is an antineoplastic agent widely used to treat several solid tumor types. The primary mechanism of action of paclitaxel is based on microtubule stabilization inducing cell-cycle arrest. Here, we use several tumor models to show that paclitaxel not only induces tumor cell-cycle arrest, but also promotes antitumor immunity. In vitro, paclitaxel reprogrammed M2-polarized macrophages to the M1-like phenotype in a TLR4-dependent manner, similarly to LPS. Paclitaxel also modulated the tumor-associated macrophage (TAM) profile in mouse models of breast and melanoma tumors; gene expression analysis showed that paclitaxel altered the M2-like signature of TAMs toward an M1-like profile. In mice selectively lacking TLR4 on myeloid cells, for example, macrophages (LysM-Cre+/-/TLR4fl/fl), the antitumor effect of paclitaxel was attenuated. Gene expression analysis of tumor samples from patients with ovarian cancer before and after treatment with paclitaxel detected an enrichment of genes linked to the M1 macrophage activation profile (IFNγ-stimulated macrophages). These findings indicate that paclitaxel skews TAMs toward an immunocompetent profile via TLR4, which might contribute to the antitumor effect of paclitaxel and provide a rationale for new combination regimens comprising paclitaxel and immunotherapies as an anticancer treatment.Significance: This study provides new evidence that the antitumor effect of paclitaxel occurs in part via reactivation of the immune response against cancer, guiding tumor-associated macrophages toward the M1-like antitumor phenotype.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5891/F1.large.jpg Cancer Res; 78(20); 5891-900. ©2018 AACR See related commentary by Garassino et al., p. 5729.

MiR-150 enhances apoptotic and anti-tumor effects of paclitaxel in paclitaxel-resistant ovarian cancer cells by targeting Notch3.

Tumor recurrence by obtaining chemoresistance is a major obstacle to treating ovarian cancer. By TargetScan database and a luciferase reporter assay, we identified miR-150 directly targets Notch3, which is a key oncogene in ovarian cancer. We, therefore, investigated the role of miR-150 in ovarian cancer cells, and the usefulness of miR-150 as a therapeutic target in chemoresistant ovarian cancer, through examining miR-150 expression by qRT-PCR in ovarian cancer cell lines and tissues, and assessing the gain-of-function effect by WST, colony forming, TUNEL, wound healing and angiogenesis assays. Western blotting was performed to evaluate its downstream targets.The miR-150 expression was significantly downregulated in ovarian cancers. Treatment with pre-miR-150 significantly inhibited cancer cell proliferation, and induced apoptosis in PTX (paclitaxel) -resistant SKpac cells, which was not seen by PTX only treatment. On spheroid forming assay, an additional pre-miR-150 treatment with PTX decreased cancer stem cell activation in PTX-resistant SKpac cells. An experimental upregulation of miR-150 also decreased cancer cell migration and angiogenesis in SKpac cells. The Notch3 downstream proteins(NICD3 and HEY2), and cell cycle-related proteins (cyclinD3, pS6, and NF-kB), and apoptosis-related proteins (BCL-2 and BCL-W) were significantly downregulated by pre-miR-150 transfection.Taken together, miR-150 is related with PTX-resistance in ovarian cancer, and treatment with pre-miR-150 resensitizes cancer cells to PTX. Therefore, it may be a promising treatment strategy in chemoresistant and recurrent ovarian cancer.

Sa1456 - The Anti-Tumor Effect of Paclitaxel, Gemcitabine and Mitomycin C Eluting Membrane in Animal Model

Sa1456 - The Anti-Tumor Effect of Paclitaxel, Gemcitabine and Mitomycin C Eluting Membrane in Animal Model

Preparation and Drug Release Properties of a Novel PLA-PEG-PTX Pro-Drug

Lactide (LA) and PEG as raw materials, 2,2-Dimethylol Propionic Acid (DMPA) as initiator, hexamethylene diisocyanate (HDI) as coupling agent, amphiphilic block copolymer PLA-PEG with side carboxyl group was synthesized. A novel PLA-PEG-PTX pro-drug was prepared through the esterification reaction between the side carboxyl group on copolymer and hydroxyl group on paclitaxel. Its structure was characterized with 1H-NMR and FT-IR. The pro-drug micelles were prepared by solvent evaporation method. The particle size of the micelles was determined by nanoparticle tracking analyzer, the micro structure of the micelles was determined by TEM, and the drug release properties were investigated by dialysis experiments. Results showed that the pro-drug micelles are spherical with core-corona structure and major particles size in 55-85nm. The PLA-PEG750-PTX (molar ratio of LA to DMPA as 40 to 1) pro-drug micelles released paclitaxel slowly and steadily without obvious burst release in buffer solution with pH=7.0, and its cumulative release rate reached to 36% in 10 hours. PLA-PEG750-PTX pro-drug micelles have a certain sustained-release effect, which is beneficial to improve the anti-tumor effect of paclitaxel.

Evaluation of Paclitaxel Effects in The Pattern of Expression of Survival and Apoptotic Genes Regulators in HeLa Cells.

We aim to determine the regulation of apoptosis by paclitaxel-induced and understand cancer dynamics to treatment targets for HeLa cells by identifying decrease/increase genes expression on HeLa cells. In this study, the anti-tumor effects of Paclitaxel (PAC) on HeLa cells have been studied in order to determine the cellular and molecular mechanisms of these effects. PAC has been applied to HeLa cells in 6 different doses (3, 7.5, 15, 30, 60, 120 nM) for 48 hours and the IC50 dose MTT method, has been determined with apoptic index (AI) DAPI. Morphological aspects have been demonstrated using light, phase contrast and fluorescent microscopes, additionally activation of Caspase 3,7 and 10 have been shown using florescent spectroscopy. RT-PCR and qRT-PCR have been used to evaluate pro/anti-apoptotic gene expression. According to the parameters being evaluated; PAC has reduced cell multiplication based on dosage and time (p<0.01). 15 nM has been determined as the IC50 value. AI value has been determined as 42%. In the molecular level analyses in addition to the increase in Caspase3,7,10 activation, RT-PCR results show that bax, bak, bcl-x, bik, mcl-1 genes are expressed in the control group as well as the experimental 15 nM group; whereas bak, bcl-x ve bik genes have a decrease in expression compared to the control group. qRT-PCR results show that Apaf1, Bad, Bax, Bcl2L11, Caspase1, Caspase10, Caspase4, Caspase7, Dffa, Fas, Htra2, Lrdd, NFKB1, NFKB2, PMAIP1, RELA, RELB, TNFRSF10A, TNFRSF10C, TNFRSF10D, TNFRSF1A, TNFRSF21, TNFRSF25 gene expressions have increased significantly. On the other hand, BAG1, BBC3, Bcl2L1, Bcl2L10, Bid, Caspase2, Caspase6, Caspase8, Caspase9, FADD, FAM96A, FasLG, HRK, SOCS3, TNF, TNFSF10, TRAF5, TRAF6 mRNA levels are significantly decreased.

Paclitaxel suppresses the viability of breast tumor MCF7 cells through the regulation of EF1α and FOXO3a by AMPK signaling.

Paclitaxel (Taxol), a potent drug of natural origin isolated from the bark of the Pacific yew, is widely used for treating ovarian, lung and breast cancers. Currently, there is little information regarding the specific mechanism underlying the anticancer activity of paclitaxel. In the present study, we found that 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR), a well-known activator of adenosine monophosphate (AMP)-activated protein kinase (AMPK), downregulated the protein and mRNA levels of elongation factor 1 α (EF1α) in breast cancer MCF7 cells. Paclitaxel increased the phosphorylation of AMPK and also downregulated the expression of EF1α in MCF7 cells. In addition, paclitaxel increased the expression, as well as the phosphorylation of forkhead box O3a (FOXO3a). Phosphorylation of FOXO3a was suppressed in the presence of compound C, a specific AMPK inhibitor, suggesting the involvement of AMPK in paclitaxel-induced FOXO3a phosphorylation. The induction and phosphorylation of FOXO3a by paclitaxel were not observed in EF1α and AMPK knockdown cells. Co-treatment with AICAR resulted in increased susceptibility of cancer cells to paclitaxel-induced suppression of their viability and further enhanced paclitaxel-induced FOXO3a phosphorylation. These results suggest that the antitumor effects of paclitaxel in breast cancer are mediated by activation of the AMPK/EF1α/FOXO3a signaling pathway.

Plk1‐dependent microtubule dynamics promotes androgen receptor signaling in prostate cancer

The androgen receptor (AR) signaling continues to be essential in castrate-resistant prostate cancer (CRPC). Taxel-based chemotherapy is the current standard treatment for CRPC patients. Unfortunately, almost all patients eventually develop resistance toward this chemotherapy. Significantly, it was recently found that the anti-tumor effect of paclitaxel in CRPC is due to its inhibition of AR activity via its inhibition of microtubule dynamics. Polo-like kinase 1 (Plk1), a critical regulator in many cell cycle events, is elevated in prostate cancer (PCa) and linked to tumor grades. Of note, we have previously shown that Plk1 phosphorylates CLIP-170 and p150(Glued) , two important regulators of microtubule dynamics. We compared paclitaxel-mediated phenotypes (inhibition of the AR signaling, decrease of microtubule dynamics and cell death) of PCa cells expressing different forms of CLIP-170 and p150(Glued) with different Plk1 phosphorylation states. We show that Plk1 phosphorylation of CLIP-170 and p150(Glued) affects cellular responses to paclitaxel. Expression of Plk1-unphosphorylatable mutants of CLIP-170 and p150(Glued) results in increased paclitaxel-induced apoptosis, increased protein degradation of the AR, and decreased nuclear accumulation of the AR in response to androgen in prostate cancer cells. Finally, we show that cells expressing unphosphorylatable mutants of CLIP-170 have defective microtubule dynamics, thus providing a new mechanism to understand how Plk1-associated kinase activity promotes constitutive activation of AR signaling in CRPC. Our data suggest that a combination of inhibition of Plk1 and paclitaxel might be a novel avenue for treatment of CRPC.

Abstract 237: Chloroquine inhibits cancer stem cells in triple negative breast cancer via regulation of DNA methylation.

Abstract Post-treatment enrichment of the tumor initiating CD44+/CD24- breast cancer stem cell (CSC) population is believed to be responsible for breast tumor recurrence and metastasis. Among breast cancer subtypes, triple negative breast cancer (TNBC) particularly is known to have an abundant CSC population, and is characterized by frequent metastatic recurrence. Chloroquine (CQ), an anti-malarial drug, is a lysotropic reagent that inhibits autophagy. Recently, CQ has been shown to reduce the CSC population in various cancers and has been tested in clinical studies. Concurrently, CQ was identified as a potential CSC inhibitor discovered from gene expression signatures of the CD44+/CD24- CSC population. However, little, aside from inhibition of autophagy, is known about the working mechanism of chloroquine in reducing CSCs, particularly in TNBCs. Based on recent recommendations that low doses of CQ be used to limit toxicities in the heart and retina, we investigated how low doses of CQ enhance the antitumor effects of paclitaxel (PTX) and reduce the CSC population. CSC population changes and apoptosis were analyzed using flow cytometry analysis (CSC: CD44+/CD24-; apoptosis: annexin V) and western blot analysis for cleaved caspase 3, and CSC function was measured by mammosphere formation efficiency in SUM159PT, MDA-MB-231, MDA-MB-468, and Hs578T TNBC cell lines. We observed enhanced cytotoxic effects and significant reduction of the CSC population by combined treatment of PTX and CQ compared to single treatment of either PTX (5nM) or CQ (1 or 5 uM) in SUM159, Hs578T, and MDA-MB-231 cells. The enhanced cytotoxicity by co-treatment of CQ and PTX correlated well with inhibition of autophagy, as indicated by cleavage of LC3B, increased expression of p62, and accumulation of autophagosomes. Moreover, the combined treatment inhibited PTX induced STAT3 activation in CSCs and epigenetically regulated gene expression critical in maintenance of CSCs via repression of DNMT1 expression. Finally, we observed enhanced therapeutic efficacy in vivo with the combination of CQ (10-20 mg/kg, daily) and PTX (30 mg/kg, two times per week) when compared to either CQ or PTX alone (p&amp;lt; 0.05). Herein, we demonstrate effective reduction of the CD44+/CD24- CSC population by combined CQ and PTX treatment through autophagy inhibition. Moreover, we found that the low-dose combined treatment of CQ with PTX was able to regulate gene expression by altering DNA methylation, subsequently reducing CSCs in the TNBC cancer cells. Thus, a low dose treatment of CQ along with chemotherapy may be effective in treating TNBC patients, lend further support for in-depth studies on the mechanism of CQ among the subgroups of TNBC. Citation Format: Dong Soon Choi, Elvin Blanco, Sergio M. Granados-Principal, Bhuvanesh Dave, Melissa Landis, Helen Wong, Jenny Chang. Chloroquine inhibits cancer stem cells in triple negative breast cancer via regulation of DNA methylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 237. doi:10.1158/1538-7445.AM2013-237

Prevention of paclitaxel‐induced peripheral neuropathy by lithium pretreatment

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect that occurs in many patients undergoing chemotherapy. It is often irreversible and frequently leads to early termination of treatment. In this study, we have identified two compounds, lithium and ibudilast, that when administered as a single prophylactic injection prior to paclitaxel treatment, prevent the development of CIPN in mice at the sensory-motor and cellular level. The prevention of neuropathy was not observed in paclitaxel-treated mice that were only prophylactically treated with a vehicle injection. The coadministration of lithium with paclitaxel also allows for administration of higher doses of paclitaxel (survival increases by 60%), protects against paclitaxel-induced cardiac abnormalities, and, notably, does not interfere with the antitumor effects of paclitaxel. Moreover, we have determined a mechanism by which CIPN develops and have discovered that lithium and ibudilast inhibit development of peripheral neuropathy by disrupting the interaction between paclitaxel, neuronal calcium sensor 1 (NCS-1), and the inositol 1,4,5-trisphosphate receptor (InsP3R) to prevent treatment-induced decreases in intracellular calcium signaling. This study shows that lithium and ibudilast are candidate therapeutics for the prevention of paclitaxel-induced neuropathy and could enable patients to tolerate more aggressive treatment regimens.

Abstract 1785: MET/VEGFR dual inhibition and prominent safety profile of TAS-115 are favorable for the combination with chemotherapeutic drugs

Abstract MET is known as a proto-oncogene, which involves in growth, migration, metastasis and angiogenesis in tumor progression. Recently, it is reported that MET also functions as chemo and EGFR-TKI resistant factor in cancer cells. Particularly, HGF/MET signal seemed to reduce the potency of conventional chemotherapeutic drugs in clinical. Meanwhile, VEGF/VEGFR signal plays an important role in angiogenesis in tumor growth, and is essential for the survival signal of endothelial cells(ECs) to protect from the damage by chemotherapeutic drugs. Some VEGFR inhibitors have already been approved for the treatment of several carcinomas with monotherapy. Many clinical combination trials with VEGFR inhibitor and chemotherapeutic agents have been tried, but its therapeutic benefit are restricted. It is highly desire for improving therapeutic benefit by combination therapy with VEGFR inhibitor and standard chemotherapy. TAS-115, a novel oral MET/VEGFR dual inhibitor, demonstrated not only potent efficacy but also prominent safety profiles achieved by improvement of PK profile and cellular selectivity. Potent MET/VEGFR dual inhibition provides significant anti-tumor effect from lower dose for TAS-115. On the one hand, the improvement of PK profile and cell selectivity led to better tolerability. As a result, therapeutic window (MTD/ED50) of TAS-115 became wider than that of the pre-existing VEGFR or MET/VEGFR inhibitors. These profiles give TAS-115 the long-term dosing at full effective dose without severe toxicity. Moreover, in vitro study, HGF completely suppressed the induction of apoptosis by 5-FU or Paclitaxel in cancer cells expressed MET. Meanwhile, VEGF abolished the cytotoxic effect by these chemotherapeutic drugs against ECs. It was suggested that harmonization of both HGF and VEGF in tumor greatly contributed to the resistance against conventional chemotherapeutic drugs. The MET/VEGFR dual inhibition by TAS-115 improves the sensitivity of chemotherapeutic drugs for both cancer cells and ECs via suppression of function as chemo-resistance factor by MET and VEGFR. In fact, the combination therapies with TAS-115 and conventional chemotherapeutic drugs caused tumor regression or potent growth inhibition without sever body weight loss in several xenograft models. Notably, TAS-115 markedly enhanced anti-tumor effect of Paclitaxel, and brought tumor shrinkage of approximately 80% in the combination. More important feature is that TAS-115 has disturbed rapid tumor re-growth following Paclitaxel treatment and the higher safety profiles of TAS-115 permits combination therapy with chemotherapeutic drugs in chronic treatment. It is expected TAS-115 demonstrates prominent therapeutic benefit in combination with chemotherapy based on the synergistic action of the potent MET/VEGFR dual inhibition and higher safety profiles. 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 1785. doi:1538-7445.AM2012-1785

Paclitaxel-eluting covered metal stents versus covered metal stents for distal malignant biliary obstruction: a prospective comparative pilot study

In unresectable malignant biliary obstruction, stent insertion can be the treatment of choice. However, metal stents are susceptible to occlusion by tumor ingrowth or overgrowth. The recently introduced paclitaxel-eluting covered metal stents (PECMSs) may prevent occlusion from tumor ingrowth by the antitumor effect of paclitaxel. Our goal was to compare the efficacy and complication rates of PECMSs and the control covered metal stents (CCMSs) in patients with malignant biliary obstruction. Prospective, randomized pilot study. Single tertiary referral center. From November 2006 to June 2008, 52 patients with unresectable distal malignant biliary obstruction were enrolled. A PECMS was inserted in 26 patients and a CCMS in 26 patients. Of these patients, 2 patients in the PECMS group and 1 patient in the CCMS group were excluded. Finally 49 patients (24 patients in the PECMS group and 25 patients in the CCMS group) were included in the analysis. PECMSs and CCMSs were inserted by using the standard ERCP technique. The mean follow-up period was 194.0 ± 144.2 days in the PECMS group and 238.8 ± 147.4 days in the CCMS group (P = .287). The stent patency duration and survival time were not significantly different between the 2 groups (P = .307 and P = .596, respectively). Stent occlusion caused by tumor ingrowth occurred in 5 patients in the PECMS group. In the CCMS group, stent occlusion caused by tumor ingrowth (n = 4) or distal stent migration (n = 4) occurred in 8 patients. In the PECMS group, 3 cases of transient cholangitis-like symptoms without stent occlusion and 1 case of pancreatitis occurred after stent insertion. One patient in the CCMS group experienced pancreatitis. This study was a pilot study in which no power calculation was done. It was performed at a single center and was performed in a nonblinded fashion. PECMSs may be safe with acceptable complication rates. There were no significant differences in the duration of stent patency and patient survival time between PECMSs and CCMSs in patients with distal malignant biliary obstruction. However, a larger study powered to detect differences must be done to conclude that there is any benefit of PECMSs.

Evaluation of the effect of calpain inhibitor AK295 on paclitaxel-induced neuropathy and antitumor activity in human cancer xenograft models.

e13510 Background: Paclitaxel is a chemotherapeutic agent that induces apoptosis of tumor cells in several cancer types including ovarian and breast cancer. However, the neurotoxic side effects of paclitaxel limit its therapeutic potential. Paclitaxel treatment has been shown to produce changes in the mitochondrial membrane-potential to increase intracellular Ca2+ causing the activation of calpain. AK295 is a calpain inhibitor which prevents the peripheral neuropathy induced by paclitaxel. The objective of this study is to find out whether or not AK295 prevents paclitaxel-induced neuropathy without affecting the antitumor activity of paclitaxel in human xenograft models. Methods: Immunocompromized mice bearing subcutaneous tumors (MDA-MB-231 and Ovcar3) (n=8/group) were treated with paclitaxel (20mg/kg, every 4th day for 3 weeks, i.v.) and AK295 (24mg/kg/day, oral or Alzet pump for 14 days), as single agent and in combination. Results: The data showed (a) AK295 alone had no effect on the growth of the tumors, (b) treatment with AK295 does not interfere with the antitumor effect of paclitaxel, and (c) the combination of paclitaxel and AK295 was surprisingly more effective (tumor growth inhibition: 51.7% on day 14) in the breast cancer model compared to paclitaxel alone (40.9% on day 20). In terms of neuropathy, MS Wang et al (Brain 2004; 127, 671) demonstrated complete protection (paclitaxel + AK295) versus ∼50% (paclitaxel alone) in tail sensory nerve action potential. Similarly, complete protection (paclitaxel + AK295) versus ∼60% (paclitaxel alone) was observed for neuromuscular function when tested on a Rotarod apparatus. Conclusions: These results demonstrate for the first time a significant improvement in the therapeutic index (complete neurotoxicity prevention with enhancement of antitumor potency) of paclitaxel in human xenograft models. No significant financial relationships to disclose.

The Anti-Tumor Effect of Motesanib in Pre-Clinical Models of Human Basal-Like Breast Cancer.

Abstract Background: Growth of breast cancer is dependent upon sustained angiogenesis, of which vascular endothelial growth factor (VEGF) is a major stimulant. Increased expression of VEGF in breast cancers is associated with poor prognosis and disease recurrence, and anti-angiogenic agents are being developed as potential new therapies. Motesanib is an oral inhibitor of VEGF receptors 1, 2 and 3, platelet-derived growth factor receptor, and stem cell factor receptor (c-Kit). This study determined the effects of motesanib on the growth of human basal-like breast cancer cell lines in immunodeficient mice, alone and in combination with paclitaxel.Materials and Methods: Breast cancer cells BT-549 and GILM2 were injected into the mammary fatpad (MFP) of mice; HCC1187 cells were injected on the subcutaneous flank. Mice with established tumors (150-200 mm3) received motesanib orally BID, at 7.5, 25 or 75 mg/kg for three weeks. Tumors were collected for analysis of blood vessels (CD-31 staining), viable tumor area, and for markers of proliferation (Ki67) and apoptosis (cleaved caspase-3). GILM2 cells were injected into the tibia of mice to model growth in bone; mice with GILM2 MFP or tibia tumors received motesanib (25 mg/kg BID) or paclitaxel (24 mg/kg Q4Dx5), or the combination. Intratibial GILM2 tumors expressing luciferase were monitored by bioluminescence and radiography to evaluate osteolysis. Analyses of tumor size were assessed by ANOVA, and immunohistochemical tests by Student's t tests.Results: Motesanib produced dose-dependent inhibition of growth of BT-549, GILM2 and HCC1187 tumors. All doses of motesanib significantly inhibited the growth of BT-549 tumors (&amp;gt;55% inhibition, P&amp;lt;0.01 vs. control). For GILM2 tumors, 25 and 75 mg/kg motesanib gave 47% and 58% growth inhibition, respectively (P&amp;lt;0.008), and for HCC1187, these doses produced 81% and 91% growth inhibition, respectively (P&amp;lt;0.001). GILM2 tumors from mice receiving &amp;gt;25mg/kg had smaller viable tissue areas (P&amp;lt;0.001), and fewer CD-31 positive blood vessels (P&amp;lt;0.001) than tumors from control mice. Immunostaining showed that 25mg/kg motesanib reduced proliferation of GILM2 tumors (P&amp;lt;0.001 vs. control), with no change in numbers of apoptotic cells. Treatment with 25 mg/kg motesanib or paclitaxel (24mg/kg Q4Dx5) significantly reduced GILM2 tumor burden in the MFP (46% and 49% inhibition, respectively) and in the bone (77% and 68% inhibition, respectively) (P&amp;lt;0.05 vs. control). Combining the agents did not enhance the anti-tumor effects of paclitaxel. Bioluminescent measurements and X-rays showed that motesanib alone was as effective as paclitaxel at reducing GILM2 growth and osteolytic destruction in bone (P&amp;lt;0.05, vs. control).Conclusion: Oral administration of motesanib significantly inhibited the growth of three ER-, PR- HER2-negative basal-like breast cancer cell lines in vivo. Motesanib was as effective as paclitaxel at controlling the growth of GILM2 in the MFP, and reduced osteolysis in bone tumors. These data suggest that motesanib has anti-angiogenic action in diverse tumor microenvironments. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5057.

다층 배양된 암세포에서 파크리탁셀의 항증식효과 분석

Human solid tumors exhibit a multicellular resistance (MCR) resulting from limited drug penetration and decreased sensitivity of tumor cells when interacting with their microenvironments. Multicellular cultures represent solid tumor condition in vivo and provide clinically relevant data. There is little data on antitumor effect of paclitaxel (PTX) in multicellular cultures although its MCR has been demonstrated. In the present study, we evaluated antiproliferative effects of PTX in multicellular layers (MCL) of DLD-1 human colorectal carcinoma cells. BrdU labeling index (LI), thickness of MCL, cell cycle distribution and cellular uptake of calcein were measured before and after exposure to PTX at 0.1 to 50 for 24, 48 and 72 hrs. BrdU LI and thickness of MCL showed a concentration- and time-dependent decrease and the changes in both parameters were similar, i.e., 34.2% and 40.6% decrease in BrdU LI and thickness, respectively, when exposed to for 72 hr. The DLD-1 cells grown in MCL showed increase in and resistance to cell cycle arrest and apoptosis compared to monolayers. Calcein uptake in MCL did not change upon PTX exposure, indicating technical problems in multicellular system. Overall, these data indicate that antitumor activity of PTX may be limited in human solid tumors (a multicellular system) and MCL may be an appropriate model to study further pharmacodynamics of PTX.