Cytotoxic Effects Of Chemotherapeutic Agents Research Articles

IMPDH Inhibition Decreases TERT Expression and Synergizes the Cytotoxic Effect of Chemotherapeutic Agents in Glioblastoma Cells.

IMP dehydrogenase (IMPDH) inhibition has emerged as a new target therapy for glioblastoma multiforme (GBM), which remains one of the most refractory tumors to date. TCGA analyses revealed distinct expression profiles of IMPDH isoenzymes in various subtypes of GBM and low-grade glioma (LGG). To dissect the mechanism(s) underlying the anti-tumor effect of IMPDH inhibition in adult GBM, we investigated how mycophenolic acid (MPA, an IMPDH inhibitor) treatment affected key oncogenic drivers in glioblastoma cells. Our results showed that MPA decreased the expression of telomerase reverse transcriptase (TERT) in both U87 and U251 cells, and the expression of O6-methylguanine-DNA methyltransferase (MGMT) in U251 cells. In support, MPA treatment reduced the amount of telomere repeats in U87 and U251 cells. TERT downregulation by MPA was associated with a significant decrease in c-Myc (a TERT transcription activator) in U87 but not U251 cells, and a dose-dependent increase in p53 and CCCTC-binding factor (CTCF) (TERT repressors) in both U87 and U251 cells. In U251 cells, MPA displayed strong cytotoxic synergy with BCNU and moderate synergy with irinotecan, oxaliplatin, paclitaxel, or temozolomide (TMZ). In U87 cells, MPA displayed strong cytotoxic synergy with all except TMZ, acting primarily through the apoptotic pathway. Our work expands the mechanistic potential of IMPDH inhibition to TERT/telomere regulation and reveals a synthetic lethality between MPA and anti-GBM drugs.

Enhancing the Impact of Chemotherapy on Ewing Sarcoma Cells through Combination with Cold Physical Plasma.

Although Ewing's sarcoma (ES) is a rare, but very aggressive tumor disease affecting the musculoskeletal system, especially in children, it is very aggressive and difficult to treat. Although medical advances and the establishment of chemotherapy represent a turning point in the treatment of ES, resistance to chemotherapy, and its side effects, continue to be problems. New treatment methods such as the application of cold physical plasma (CPP) are considered potential supporting tools since CPP is an exogenous source of reactive oxygen and nitrogen species, which have similar mechanisms of action in the tumor cells as chemotherapy. This study aims to investigate the synergistic effects of CPP and commonly used cytostatic chemotherapeutics on ES cells. The chemotherapy drugs doxorubicin and vincristine, the most commonly used in the treatment of ES, were applied to two different ES cell lines (RD-ES and A673) and their IC20 and IC50 were determined. In addition, individual chemotherapeutics in combination with CPP were applied to the ES cells and the effects on cell growth, cell viability, and apoptosis processes were examined. A single CPP treatment resulted in the dose-dependent growth inhibition of ES cells. The combination of different cytostatics and CPP led to significant growth inhibition, a reduction in cell viability, and higher rates of apoptosis compared to cells not additionally exposed to CPP. The combination of CPP treatment and the application of cytostatic drugs to ES cells showed promising results, significantly enhancing the cytotoxic effects of chemotherapeutic agents. These preclinical in vitro data indicate that the use of CPP can enhance the efficacy of common cytostatic chemotherapeutics, and thus support the translation of CPP as an anti-tumor therapy in clinical routine.

A phase 1b study of andecaliximab in combination with S-1 plus platinum in Japanese patients with gastric adenocarcinoma

Andecaliximab (ADX) is a monoclonal antibody that inhibits matrix metalloproteinase 9 (MMP9), an extracellular enzyme involved in matrix remodeling, tumor growth, and metastasis. In preclinical models, MMP9 inhibitors have been shown to enhance the cytotoxic effects of chemotherapeutic agents and to suppress distant metastasis. In this phase Ib, multicenter study, the safety and efficacy of ADX combined with S-1 plus cisplatin (SP) or S-1 plus oxaliplatin (SOX) as a first-line treatment were evaluated in Japanese patients with advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma. ADX was administrated at a dose of 800 mg every 2 weeks for the SP cohort and 1200 mg every three weeks for the SOX cohort. As of December 2019, 16 patients were enrolled (six patients in the SP cohort and 10 patients in the SOX cohort). Peripheral sensory neuropathy (69%), anorexia (63%), nausea (56%), and decreased neutrophil counts (44%) were the most common adverse events (AEs). The grade 3 or higher AEs attributed to ADX were stomatitis and abnormal hepatic function (each one patient) in the SP cohort and decreased neutrophil counts (two patients) in the SOX cohort. The objective response rate in 11 patients with measurable target lesions was 73% (8/11), based on the investigator’s evaluation. Median progression-free survival was11.9 months (90% confidence interval, 5.6–16.6), and median overall survival was not reached. In conclusion, ADX combined with S-1 plus platinum demonstrated a manageable safety profile and promising clinical activity in the first-line treatment of patients with advanced gastric or GEJ adenocarcinoma.Clinical Trial Registration information: ClinicalTrials.gov Identifier: NCT02862535 (11/08/2016) and protocol ID: GS-US-296-1884.

Blockade of ALDH in Cisplatin-Resistant Ovarian Cancer Stem Cells In Vitro Synergistically Enhances Chemotherapy-Induced Cell Death.

Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer-related death. The high mortality and morbidity associated with EOC are mostly due to late diagnosis and chemotherapy drug resistance. Currently, the standard first-line chemotherapy regimen is systemic administration of platinum-based chemotherapy combined with a taxane. A major problem besides cisplatin resistance (occurring in nearly one-third of patients) is the greater toxicity of the drug combinations. A synergistic treatment with drug supporting activity could maximize the cytotoxic effects of chemotherapeutic agents on tumor cells while decreasing the dosage of each drug to potentially reduce toxicity. The ALDH-blocking agent Disulfiram (DSF), a clinically approved drug used for alcoholism treatment, has displayed promising anti-cancer activity. We previously described that blocking ALDH activity enhances the induction of apoptosis, especially in ovarian cancer stem cells treated with chemotherapeutic agents. In this study, we further investigated the synergistic effect of DSF in combination with cytotoxic chemotherapeutic drugs. The concentration of each chemotherapeutic agent could be significantly reduced with sustained efficacy on tumor cell apoptosis in cell lines in vitro (Dose-Reduction Index at IC50 from 1 to 50). Moreover, the potential chemo-sensitizing effects of DSF on ALDH-associated cisplatin-resistant ovarian cancer stem cells were also investigated and shown that in contrast to its high resistance to cisplatin, the cisplatin-resistant cells remain very sensitive to DSF-induced cytotoxicity (apoptosis and necrosis: cisplatin-resistant cells vs. parental cells: 60.4% vs. 20.5%). In combination with DSF and cisplatin, relatively more apoptosis and necrosis were induced in cisplatin-resistant cells than in their parental cells (apoptosis and necrosis: cisplatin-resistant cells vs. parental cells: 81.5% vs. 50.1%). A transcriptome analysis identified that ALDH was mainly enriched in the cancer-associated fibroblasts and showed that ALDH plays roles in responding to oxidative stress, metabolisms, and energy transition in the ALDH-associated cisplatin-resistant ovarian cancer stem cells. In conclusion, our data demonstrate a key role of ALDH-associated cisplatin-resistant cancer stem cells and identifies DSF as a potential adjuvant for a rational protocol design by computational quantitative assessment in vitro on ovarian cancer cell lines. Our work contributes to resolving the ALDH-associated cisplatin resistance and provides a resource for the development of novel chemotherapeutic regimens.

The Potential Role of Sildenafil in Cancer Management through EPR Augmentation.

Enhanced permeation retention (EPR) was a significant milestone discovery by Maeda et al. paving the path for the emerging field of nanomedicine to become a powerful tool in the fight against cancer. Sildenafil is a potent inhibitor of phosphodiesterase 5 (PDE-5) used for the treatment of erectile dysfunction (ED) through the relaxation of smooth muscles and the modulation of vascular endothelial permeability. Overexpression of PDE-5 has been reported in lung, colon, metastatic breast cancers, and bladder squamous carcinoma. Moreover, sildenafil has been reported to increase the sensitivity of tumor cells of different origins to the cytotoxic effect of chemotherapeutic agents with augmented apoptosis mediated through inducing the downregulation of Bcl-xL and FAP-1 expression, enhancing reactive oxygen species (ROS) generation, phosphorylating BAD and Bcl-2, upregulating caspase-3,8,9 activities, and blocking cells at G0/G1 cell cycle phase. Sildenafil has also demonstrated inhibitory effects on the efflux activity of ATP-binding cassette (ABC) transporters such as ABCC4, ABCC5, ABCB1, and ABCG2, ultimately reversing multidrug resistance. Accordingly, there has been a growing interest in using sildenafil as monotherapy or chemoadjuvant in EPR augmentation and management of different types of cancer. In this review, we critically examine the basic molecular mechanism of sildenafil related to cancer biology and discuss the overall potential of sildenafil in enhancing EPR-based anticancer drug delivery, pointing to the outcomes of the most important related preclinical and clinical studies.

Is platelet-rich plasma improves the anastomotic healing in hyperthermic intraperitoneal chemotherapy with oxaliplatin: an experimental rat study.

PurposeHyperthermic intraperitoneal chemotherapy (HIPEC) is a novel treatment option for peritoneal surface malignancies. Due to cytotoxic effects of chemotherapeutic agents, anastomosis healing can be impaired and lead to leakage rates higher than conventional intestinal surgery. In this experimental study, we aimed to investigate the effects of platelet-rich plasma (PRP) on colonic anastomosis in rats that received HIPEC with oxaliplatin.MethodsThirty rats were divided into 3 groups. Group 1 was determined as control group and hyperthermic saline perfusion was performed after colon anastomosis. In group 2, colon anastomosis then hyperthermic oxaliplatin perfusion was performed. In the last group, the colonic anastomosis was enhanced by PRP gel and then hyperthermic oxaliplatin perfusion was performed. All the rats were reoperated on postoperative day 7 and anastomotic bursting pressure values were recorded. Tissue samples were taken for hydroxyproline assay and histopathological examination.ResultsControl group had higher anastomotic bursting pressure value than group 2 and group 3 (P < 0.001). There were significant differences in anastomotic bursting pressure between groups 2 and 3 (P < 0.001). Group 2 had significantly lower hydroxyproline levels than group 3 and control group (P < 0.001). Histopathological examination revealed that PRP application reduced inflammatory response.ConclusionPRP application on colonic anastomosis improves anastomotic healing and can reduce anastomosis related complications and stoma creation; though further clinical studies are needed.

The role of Clusterin in cancer metastasis.

Clusterin is a conserved glycoprotein that has been characterized from almost all human tissues and fluids and plays a key role in cellular stress response and survival. Recently, research efforts have been contributed to explore the function of Clusterin in cancer metastasis, which is particularly important to design the strategies for treating metastatic patients. Evidence collected has demonstrated that Clusterin is overexpressed in tumor metastatic patients and experimental metastasis models. Specifically, Clusterin has been shown to have the role in anti-apoptotic capacities, development of therapy resistance and induction of epithelial–mesenchymal transition, all associated with cancer metastasis. Inhibition of Clusterin is known to increase the cytotoxic effects of chemotherapeutic agents and improves advanced cancer patients survival in clinical trials. Our unpublished data have demonstrated that Clusterin is overexpressed in bladder cancer and metformin, a well-known metabolism modulator specifically targets Clusterin by inhibiting migration of bladder cancer cells. In this review, we provide a general view of how Clusterin modulates cancer metastasis and update current understanding of detailed molecular mechanisms underlying of Clusterin for developing cancer management in future.

Analysis of Cytotoxic Effects of Chemotherapeutic Agents for Gastrointestinal Cancer with Cell-based Impedance Biosensor

Analysis of Cytotoxic Effects of Chemotherapeutic Agents for Gastrointestinal Cancer with Cell-based Impedance Biosensor

Cooling of the oral mucosa to prevent adverse effects of chemotherapeutic agents: An invitro study.

The cytotoxic effect of chemotherapeutic agents to the oral mucosa, as a side effect of cancer treatment, is a major problem. Cooling the oral mucosa using ice chips in conjunction with chemotherapy is known to reduce the severity of oral mucositis. However, although the use of ice chips is of clinical value, this method of cooling has inherent problems including discomfort for the patient, non-uniformity and fluctuations in cooling temperature throughout the oral cavity. Furthermore, despite being used clinically, it is not known what reduction in temperature is required to prevent oral mucositis. The aim of this study was therefore to determine invitro if the cytotoxic effect of 5-fluorouracil (5-FU) on the oral mucosa could be reduced by lowering the temperature during chemotherapeutic treatment. Tissue-engineered oral mucosal (TEOM) models were incubated at 20, 25, 30 or 35°C for 30minutes followed by exposure to a clinically relevant concentration of 5-FU (162μg/mL) for 2hours and compared with untreated models (35°C). Cell viability and inflammatory cytokine production (IL-6 and TNF-α) were measured using PrestoBlue® and ELISA, respectively. TEOM models incubated at 20°C showed an increased cell viability and had a reduced IL-6 and TNF-α production compared to models treated with 5-FU incubated at 35°C. This study demonstrates a reduced cytotoxic effect to the TEOM by reducing the temperature of the tissue during chemotherapy treatment and suggests that decreasing the temperature to 20°C could have clinical advantages.

Participation of MT3 melatonin receptors in the synergistic effect of melatonin on cytotoxic and apoptotic actions evoked by chemotherapeutics.

Melatonin has antitumor activity via several mechanisms including its antiproliferative and proapoptotic effects in addition to its potent antioxidant actions. Therefore, melatonin may be useful in the treatment of tumors in association with chemotherapy drugs. This study was performed to study the role of melatonin receptors on the cytotoxicity and apoptosis induced by the chemotherapeutic agents cisplatin and 5-fluorouracil in two tumor cell lines, such as human colorectal cancer HT-29 cells and cervical cancer HeLa cells. We found that both melatonin and the two chemotherapeutic agents tested induced a decrease in HT-29 and HeLa cell viability. Furthermore, melatonin significantly increased the cytotoxic effect of chemotherapeutic agents, particularly, in 5-fluorouracil-challenged cells. Stimulation of cells with either of the two chemotherapeutic agents in the presence of melatonin further increased caspase-3 activation. Concomitant treatments with melatonin and chemotherapeutic agents augmented the population of apoptotic cells compared to the treatments with chemotherapeutics alone. Blockade of MT1 and/or MT2 receptors with luzindole or 4-P-PDOT was unable to reverse the enhancing effects of melatonin on both cytotoxicity, caspase-3 activation and the amount of apoptotic cells evoked by the chemotherapeutic agents, whereas when MT3 receptors were blocked with prazosin, the synergistic effect of melatonin with chemotherapy on cytotoxicity and apoptosis was reversed. Our findings provided evidence that in vitro melatonin strongly enhances chemotherapeutic-induced cytotoxicity and apoptosis in two tumor cell lines, namely HT-29 and HeLa cells and, this potentiating effect of melatonin is mediated by MT3 receptor stimulation.

Molecular targeting of the Aurora-A/SMAD5 oncogenic axis restores chemosensitivity in human breast cancer cells.

Although the majority of breast cancers initially respond to the cytotoxic effects of chemotherapeutic agents, most breast cancer patients experience tumor relapse and ultimately die because of drug resistance. Breast cancer cells undergoing epithelial to mesenchymal transition (EMT) acquire a CD44+/CD24-/ALDH1+ cancer stem cell-like phenotype characterized by an increased capacity for tumor self-renewal, intrinsic drug resistance and high proclivity to develop distant metastases. We uncovered in human breast tumor xenografts a novel non-mitotic role of Aurora-A kinase in promoting breast cancer metastases through activation of EMT and expansion of breast tumor initiating cells (BTICs). In this study we characterized the role of the Aurora-A/SMAD5 oncogenic axis in the induction of chemoresistance. Breast cancer cells overexpressing Aurora-A showed resistance to conventional chemotherapeutic agents, while treatment with alisertib, a selective Aurora-A kinase inhibitor, restored chemosensitivity. Significantly, SMAD5 expression was required to induce chemoresistance and maintain a breast cancer stem cell-like phenotype, indicating that the Aurora-A/SMAD5 oncogenic axis promotes chemoresistance through activation of stemness signaling. Taken together, these findings identified a novel mechanism of drug resistance through aberrant activation of the non-canonical Aurora-A/SMAD5 oncogenic axis in breast cancer.

Abstract 4299: Chemosensitizing effect of telomerase-dependent oncolytic adenovirus through virally induced microRNA-29-mediated MCL1 downregulation in osteosarcoma cells

Abstract Osteosarcoma is a rare highly malignant tumor. It is generally refractory to chemotherapy, which contributes to its poor prognosis. The enhancement of chemosensitivity is a potential approach to improve the prognostic outcome of osteosarcoma patients. We developed a telomerase-specific replication-competent oncolytic adenovirus, OBP-301. We recently confirmed the antitumor effect of OBP-301 monotherapy in human osteosarcoma cells. We also revealed the synergistic chemosensitizing effect of OBP-301 in human epithelial malignant tumor cells. In this study, we investigated the chemosensitizing effect of OBP-301 and its underlying mechanisms in human osteosarcoma cells. We used four human osteosarcoma cell lines, SaOS-2, MNNG/HOS, HOS and 143B. OBP-301 is an attenuated adenovirus, in which the hTERT promoter drives the expression of E1 gene, and causes tumor-selective lysis in a variety of human malignant tumor cells with telomerase activity. OBP-301 infection enhanced the cytotoxic effect of chemotherapeutic agents (cisplatin and doxorubicin). The calculation of combination index revealed the synergistic effects in all four human osteosarcoma cell lines. Combination of OBP-301 increased chemotherapy-induced apoptosis. To clarify the molecular mechanism underlying the chemosensitizing effect of OBP-301, we investigated whether OBP-301 affects the expression of anti-apoptotic BCL2 family proteins. In SaOS-2 and MNNG/HOS cells, the expression level of MCL1 was markedly decreased by OBP-301 infection. MCL1 knockdown by siRNA enhanced the chemotherapy-induced apoptosis as well as OBP-301. We recently revealed that OBP-301 increase the expression of cellular miRNAs in human cancer cells. To investigate the underlying mechanism of OBP-301-mediated MCL1 suppression, we determined whether OBP-301 upregulates MCL1-targeted miRNAs. OBP-301 dose-dependently upregulated the expression of MCL1 targeted miR-29 in SaOS-2 and MNNG/HOS cells. Exogenously introduced miR-29 efficiently suppressed MCL1 expression and enhanced the chemotherapy-induced apoptosis. Moreover, combination treatment significantly inhibited tumor growth, as compared to monotherapy, in a subcutaneous xenograft tumor model. MCL1 is overexpressed in various types of human tumor cells. Overexpression of anti-apoptotic proteins is an important factor to prevent chemotherapy-induced apoptosis in cancer. We demonstrated OBP-301 efficiently kills human osteosarcoma cells and markedly sensitizes them to chemotherapy. MCL1 suppression via OBP-301-induced miR-29 is critical as the underlying mechanism of the OBP-301 mediated chemosensitizing effect. These results suggest that replicative virus-mediated tumor specific MCL1 ablation may be a promising strategy to attenuate chemoresistance in osteosarcoma patients. Citation Format: Shuhei Osaki, Hiroshi Tazawa, Joe Hasei, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Chemosensitizing effect of telomerase-dependent oncolytic adenovirus through virally induced microRNA-29-mediated MCL1 downregulation in osteosarcoma cells [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 4299. doi:10.1158/1538-7445.AM2017-4299

Re-examining HSPC1 inhibitors

HSPC1 is a critical protein in cancer development and progression, including colorectal cancer (CRC). However, clinical trial data reporting the effectiveness of HSPC1 inhibitors on several cancer types has not been as successful as predicted. Furthermore, some N-terminal inhibitors appear to be much more successful than others despite similar underlying mechanisms. This study involved the application of three N-terminal HSPC1 inhibitors, 17-DMAG, NVP-AUY922 and NVP-HSP990 on CRC cells. The effects on client protein levels over time were examined. HSPC1 inhibitors were also applied in combination with chemotherapeutic agents commonly used in CRC treatment (5-fluorouracil, oxaliplatin and irinotecan). As HSPA1A and HSPB1 have anti-apoptotic activity, gene-silencing techniques were employed to investigate the significance of these proteins in HSPC1 inhibitor and chemotherapeutic agent resistance. When comparing the action of the three HSPC1 inhibitors, there are distinct differences in the time course of important client protein degradation events. The differences between HSPC1 inhibitors were also reflected in combination treatment—17-DMAG was more effective compared with NVP-AUY922 in potentiating the cytotoxic effects of 5-fluorouracil, oxaliplatin and irinotecan. This study concludes that there are distinct differences between N-terminal HSPC1 inhibitors, despite their common mode of action. Although treatment with each of the inhibitors results in significant induction of the anti-apoptotic proteins HSPA1A and HSPB1, sensitivity to HSPC1 inhibitors is not improved by gene silencing of HSPA1A or HSPB1. HSPC1 inhibitors potentiate the cytotoxic effects of chemotherapeutic agents in CRC, and this approach is readily available to enter clinical trials. From a translational point of view, there may be great variability in sensitivity to the inhibitors between individual patients.

Uptake of lymphoma-derived exosomes by peripheral blood leukocytes.

Exosomes are nanosized lipid vesicles secreted into blood and other body fluids and serve as vehicles for intercellular communication. Despite being an important component of the tumor microenvironment (TME), exosomal targeting and uptake into recipient cells are still not fully understood. Few studies have looked at lymphoma exosomes and their interactions with circulating blood cells. In this study, we examine the exosomal uptake distribution among peripheral blood leukocytes (PBLs) using vesicles derived from a diffuse large B cell lymphoma cell line, WSU-DLCL2. Lymphoma cells survive, proliferate, and are protected from the cytotoxic effects of chemotherapeutic agents by soluble factors or by direct contact with inflammatory and stromal cells within the TME. In an attempt to close the gap in knowledge concerning lymphoma TME immunosuppression, we have treated normal human PBLs with PKH67-labeled lymphoma exosomes and monitored the uptake by measuring fluorescence at different time points using flow cytometry and fluorescent microscopy. Our results show that of the four populations examined, B cells and monocytes demonstrated uptake of PKH67-labeled exosomes, while T cells and NK cells displayed significantly less uptake.

Celecoxib enhanced the cytotoxic effect of cisplatin in drug-resistant human gastric cancer cells by inhibition of cyclooxygenase-2

Recently studies indicated that cyclooxygenase-2 might induce P-glycoprotein expression, and was involved in the development of drug resistance phenotype in human gastric cancer cells. The present study was to explore the correlation of celecoxib, a cyclooxygenase-2 specific inhibitor, and P-glycoprotein in drug-resistant gastric cancer cells. The results showed the over-expression of cyclooxygenase-2 and P-glycoprotein in cisplatin-resistant gastric cancer SGC-7901 cells (SGC-7901/DDP), suggesting the possible involvement of cyclooxygenase-2 in the development of P-glycoprotein-mediated drug resistance. Celecoxib was more effective in SGC-7901/DDP cells with a lower inhibitory concentration compared to that in SGC-7901 cells, supporting such a cyclooxygenase-2-dependent drug resistance in SGC-7901/DDP cells. Further studies revealed down-regulation of cyclooxygenase-2 and P-glycoprotein expression by celecoxib, and a decline in prostaglandin E2 release and protein kinase A level. Celecoxib-induced apoptosis of SGC-7901/DDP cells led to increased p53 expression, decreased Bcl-2/Bax ratio and up-regulated caspase-3 level. Also, celecoxib induced apoptosis in SGC-7901/DDP cells synergistically with cisplatin. Our study suggested that celecoxib might enhance the cytotoxic effect of chemotherapeutic agents in drug-resistant human gastric cancer cells through a cyclooxygenase-2-dependent manner.

Abstract 342: Telomerase-dependent oncolytic adenovirus sensitizes human osteosarcoma cells to chemotherapy through Mcl-1 downregulation

Abstract Osteosarcoma is the most common malignant primary bone tumor. Despite advances in multi-agent chemotherapy, poor response to chemotherapy is one of the critical prognostic factors in osteosarcoma. Therefore, enhancement of chemosensitivity is one approach to improve the survival of osteosarcoma patients. We recently developed a telomerase-specific replication-competent oncolytic adenovirus, Telomelysin (OBP-301). A Phase I clinical trial in the U.S. showed the safety of OBP-301 in advanced cancer patients, and a Phase I/II clinical trial for malignant thoracic tumors is currently undergoing. We recently confirmed the antitumor effect of OBP-301 monotherapy in human osteosarcoma cells. In this study, we investigated the chemosensitizing effect of OBP-301 in human osteosarcoma cells and the molecular mechanism in the OBP-301-mediated enhancement of cell death. We used four human osteosarcoma cell lines, HOS, MNNG/HOS, 143B and SaOS-2. OBP-301 is an attenuated adenovirus, in which the hTERT promoter drives the expression of E1 gene, and causes tumor-selective lysis in a variety of human malignant tumor cells with telomerase activity. OBP-301 infection enhanced the cytotoxic effect of chemotherapeutic agents, cisplatin and doxorubicin, that are commonly used for the treatment of osteosarcomas. The calculation of combination index revealed the synergistic effects in all human osteosarcoma cell lines. Combination of OBP-301 increased apoptosis in the chemotherapeutic agents-treated cells. To clarify the molecular mechanism for the chemosensitizing effect of OBP-301, the expression of Bcl-2 family proteins, which are critical regulators of apoptosis, were investigated. In OBP-301-infected MNNG/HOS and SaOS-2 cells, the expression level of Mcl-1 was markedly decreased by OBP-301 infection. Downregulation of Mcl-1 expression by siRNA enhanced the chemotherapeutic agents-induced apoptosis. Moreover, combination therapy of chemotherapeutic agents with OBP-301 significantly suppressed tumor growth in a subcutaneous xenograft tumor model compared to monotherapy with chemotherapeutic agents or OBP-301. Anti-apoptotic Mcl-1 protein has been shown to be frequently overexpressed in human sarcomas. Overexpression of anti-apoptotic proteins is an important factor to prevent chemotherapy-induced apoptosis in cancer. We demonstrated the involvement of Mcl-1 suppression in the chemosensitizing effect of OBP-301 in human osteosarcoma cells. These results suggest that Mcl-1 is a critical target for improvement of the chemosensitivity in human osteosarcoma cells and the combination of chemotherapy with OBP-301 may be a novel therapeutic strategy for osteosarcoma patients. Citation Format: Shuhei Osaki, Toshinori Omori, Hiroshi Tazawa, Joe Hasei, Yasuaki Yamakawa, Tsuyoshi Sasaki, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Telomerase-dependent oncolytic adenovirus sensitizes human osteosarcoma cells to chemotherapy through Mcl-1 downregulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2014-342

IGF-1R inhibition potentiates cytotoxic effects of chemotherapeutic agents in early stages of chemoresistant ovarian cancer cells

The kinetics and effect of hyper activated IGF-1R signaling is not well investigated during acquirement of platinum and taxol resistance in ovarian cancer cells. Herein we reported an upregulated IGF-1R expression in early stages of cisplatin paclitaxel and cisplatin–taxol resistance. Picropodophyllin, an IGF-1R inhibitor, alone and in combination with cisplatin, paclitaxel or both at lowest possible doses could reverse the resistance at early stages. Upregulated IGF-1R was also found in primary tumors of ovarian cancer patients after three to four cycles of platinum–taxol treatment. These findings indicate that a combination of cytotoxic agents and IGF-1R inhibitor is more effective at early stages of chemoresistant ovarian cancer.

CEP-33779 antagonizes ATP-binding cassette subfamily B member 1 mediated multidrug resistance by inhibiting its transport function

The overexpression of ATP-binding cassette (ABC) transporters often leads to the development of multidrug resistance (MDR), which is the major factor contributing to the failure of chemotherapy. The objective of this study was to investigate the enhancement of CEP-33779, a small-molecule inhibitor of Janus kinase 2 (JAK2), on the efficacy of conventional chemotherapeutic agents in MDR cells with overexpression of P-glycoprotein (ABCB1), multidrug resistance-associated protein 1 (ABCC1) and breast cancer resistance protein (ABCG2). Our results showed that CEP-33779, at nontoxic concentrations, significantly sensitized ABCB1 overexpressing MDR cells to its anticancer substrates. CEP-33779 significantly increased intracellular accumulation and decreased the efflux of doxorubicin by inhibiting the ABCB1 transport function. Furthermore, CEP-33779 did not alter the expression of ABCB1 both at protein and mRNA levels but did stimulate the activity of ABCB1 ATPase. CEP-33779 was predicted to bind within the large hydrophobic cavity of homology modeled ABCB1. In addition, the down-regulation of JAK2 by shRNA altered neither the expression of ABCB1 nor the cytotoxic effect of chemotherapeutic agents in ABCB1-overexpressing cells. Significantly, CEP-33779 enhanced the efficacy of vincristine against the ABCB1-overexpressing and drug resistant KBv200 cell xenograft in nude mice. In conclusion, we conclude that CEP-33779 enhances the efficacy of substrate drugs in ABCB1-overexpressing cells by directly inhibiting ABCB1 transport function. The findings encouraged to further study on the combination therapy of CEP-33779 with conventional chemotherapeutic agents in ABCB1 mediated-MDR cancer patients.

Wogonin-enhanced reactive oxygen species-induced apoptosis and potentiated cytotoxic effects of chemotherapeutic agents by suppression Nrf2-mediated signaling in HepG2 cells

Cancer cells, compared with normal cells, are under increased oxidative stress with higher level of reactive oxygen species (ROS). When exposed to environmental stresses such as ROS, NFE-related factor 2 (Nrf2) is the key to antioxidant response by transcriptionally activating various detoxification and antioxidant enzymes. Previously, we have shown that wogonin, a flavonoid isolated from the root of Scutellaria baicalensis Georgi, could reverse drug resistance in MCF-7/DOX cells by blocking the translocation of Nrf2 into nucleus. However, the exact mechanism underlying the effect remains unclear. In this study, we observed that wogonin reduced the Nrf2 nuclear translocation, and therefore elevated the level of intracellular ROS to accomplish the purpose of killing malignant cells. Furthermore, the suppression of Nrf2 by wogonin can potentiate cytotoxic effects of chemotherapeutic agents in HepG2 cells. On one hand, down-regulation of Nrf2 lead to reduction of cytoprotective effect by inducing phage II enzymes which sensitize cells to chemotherapeutic agents. On the other hand, inhibition of multidrug resistance-associated proteins (MRPs) by wogonin enhances the effective drug level in cancer cells and potentiates their chemotherapeutic effects. Finally, we found that the decrease of Nrf2 may be related to overexpression of p53. Using p53 siRNA to knock down the endogenous p53 expression, the levels of both c-myc and Nrf2 in nucleus increased when exposed to wogonin. The present study indicates that wogonin can be used in chemotherapy not only because of its own antitumor ability, but also due to the enhanced cytotoxic effects of chemotherapeutic agents.

Clusterin: a key player in cancer chemoresistance and its inhibition

Clusterin is a heterodimeric disulfide-linked glycoprotein (449 amino acids) isolated in the rat prostate after castration. It is widely distributed in different tissues and highly conserved in species. There are two isoforms (1 and 2) with antagonistic actions regarding apoptosis. Clusterin is implicated in a number of biological processes, including lipid transport, membrane recycling, cell adhesion, programmed cell death, and complement cascade, representing a truly multifunctional protein. Isoform 2 is overexpressed under cellular stress conditions and protects cells from apoptosis by impeding Bax actions on the mitochondrial membrane and exerts other protumor activities, like phosphatidylinositol 3-kinase/protein kinase B pathway activation, modulation of extracellular signal-regulated kinase 1/2 signaling and matrix metallopeptidase-9 expression, increased angiogenesis, modulation of the nuclear factor kappa B pathway, among others. Its overexpression should be considered as a nonspecific cellular response to a wide variety of tissue insults like cytotoxic chemotherapy, radiation, excess of free oxygen radicals, androgen or estrogen deprivation, etc. A review of the recent literature strongly suggests potential roles for custirsen in particular, and proapoptosis treatments in general, as novel modalities in cancer management. Inhibition of clusterin is known to increase the cytotoxic effects of chemotherapeutic agents, and custirsen, a second-generation antisense oligonucleotide that blocks clusterin, is being tested in a Phase III clinical trial after successful results were achieved in Phase II studies. A major issue in cancer evolution that remains unanswered is whether clusterin represents a driving force of tumorigenesis or a late phenomenon after chemotherapy. This review presents preclinical data that encourages trials in various types of cancer other than advanced castration-resistance prostate cancer and discusses briefly the appropriate timing for clusterin inhibition in the clinical context.