Comparative Cardiac Toxicity of Anthracyclines In Vitro and In Vivo in the Mouse

Purpose

The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo.

Methods

The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment.

Results

In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin.

Conclusion

This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.     

In Vitro and In Vivo Antitumor Activity of a Novel pH-Activated Polymeric Drug Delivery System for Doxorubicin

Background

Conventional chemotherapy agent such as doxorubicin (DOX) is of limited clinical use because of its inherently low selectivity, which can lead to systemic toxicity in normal healthy tissue.

Methods

A pH stimuli-sensitive conjugate based on polyethylene glycol (PEG) with covalently attachment doxorubicin via hydrazone bond (PEG-hyd-DOX) was prepared for tumor targeting delivery system. While PEG-DOX conjugates via amid bond (PEG-ami-DOX) was synthesized as control.

Results

The synthetic conjugates were confirmed by proton nuclear magnetic resonance (NMR) spectroscopy, the release profile of DOX from PEG-hyd-DOX was acid-liable for the hydrazone linkage between DOX and PEG, led to different intracellular uptake route; intracellular accumulation of PEG-hyd-DOX was higher than PEG-ami-DOX due to its pH-triggered profile, and thereby more cytotoxicity against MCF-7, MDA-MB-231 (breast cancer models) and HepG2 (hepatocellular carcinoma model) cell lines. Following the in vitro results, we xenografted MDA-MB-231 cell onto SCID mice, PEG-hyd-DOX showed stronger antitumor efficacy than free DOX and was tumor-targeting.

Conclusions

Results from these in vivo experiments were consistent with our in vitro results; suggested this pH-triggered PEG-hyd-DOX conjugate could target DOX to tumor tissues and release free drugs by acidic tumor environment, which would be potent in antitumor drug delivery.     

Mesenchymal stromal cells primed with paclitaxel provide a new approach for cancer therapy

Background

Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation.

Methods and Principal Findings

Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth.

Conclusions

These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.     

Downregulation of Keratin 76 Expression during Oral Carcinogenesis of Human,Hamster and Mouse

Background

Keratins are structural marker proteins with tissue specific expression; however, recent reports indicate their involvement in cancer progression. Previous study from our lab revealed deregulation of many genes related to structural molecular integrity including KRT76. Here we evaluate the role of KRT76 downregulation in oral precancer and cancer development.

Methods

We evaluated KRT76 expression by qRT-PCR in normal and tumor tissues of the oral cavity. We also analyzed K76 expression by immunohistochemistry in normal, oral precancerous lesion (OPL), oral squamous cell carcinoma (OSCC) and in hamster model of oral carcinogenesis. Further, functional implication of KRT76 loss was confirmed using KRT76-knockout (KO) mice.

Results

We observed a strong association of reduced K76 expression with increased risk of OPL and OSCC development. The buccal epithelium of DMBA treated hamsters showed a similar trend. Oral cavity of KRT76-KO mice showed preneoplastic changes in the gingivobuccal epithelium while no pathological changes were observed in KRT76 negative tissues such as tongue.

Conclusion

The present study demonstrates loss of KRT76 in oral carcinogenesis. The KRT76-KO mice data underlines the potential of KRT76 being an early event although this loss is not sufficient to drive the development of oral cancers. Thus, future studies to investigate the contributing role of KRT76 in light of other tumor driving events are warranted.     

Triterpenoids Amplify Anti-Tumoral Effects of Mistletoe Extracts on Murine B16.F10 Melanoma In Vivo

Purpose

Mistletoe extracts are often used in complementary cancer therapy although the efficacy of that therapy is controversially discussed. Approved mistletoe extracts contain mainly water soluble compounds of the mistletoe plant, i.e. mistletoe lectins. However, mistletoe also contains water-insoluble triterpenoids (mainly oleanolic acid) that have anti-tumorigenic effects. To overcome their loss in watery extracts we have solubilized mistletoe triterpenoids with cyclodextrins, thus making them available for in vivo cancer experiments.

Experimental design

B16.F10 subcutaneous melanoma bearing C57BL/6 mice were treated with new mistletoe extracts containing both water soluble compounds and solubilized triterpenoids. Tumor growth and survival was monitored. In addition, histological examinations of the tumor material and tumor surrounding tissue were performed.

Results

Addition of solubilized triterpenoids increased the anti-tumor effects of the mistletoe extracts, resulting in reduced tumor growth and prolonged survival of the mice. Histological examination of the treated tumors showed mainly tumor necrosis and some apoptotic cells with active caspase-3 and TUNEL staining. A significant decrease of CD31-positive tumor blood vessels was observed after treatment with solubilized triterpenoids and different mistletoe extracts.

Conclusion

We conclude that the addition of solubilized mistletoe triterpenoids to conventional mistletoe extracts improves the efficacy of mistletoe treatment and may represent a novel treatment option for malignant melanoma.     

RLIP76 regulates PI3K/Akt signaling and chemo-radiotherapy resistance in pancreatic cancer

Purpose

Pancreatic cancer is an aggressive malignancy with characteristic metastatic course of disease and resistance to conventional chemo-radiotherapy. RLIP76 is a multi-functional cell membrane protein that functions as a major mercapturic acid pathway transporter as well as key regulator of receptor-ligand complexes. In this regard, we investigated the significance of targeting RLIP76 on PI3K/Akt pathway and mechanisms regulating response to chemo-radiotherapy.

Research Design and Methods

Cell survival was assessed by MTT and colony forming assays. Cellular levels of proteins and phosphorylation was determined by Western blot analyses. The impact on apoptosis was determined by TUNEL assay. The anti-cancer effects of RLIP76 targeted interventions in vivo were determined using mice xenograft model of the pancreatic cancer. The regulation of doxorubicin transport and radiation sensitivity were determined by transport studies and colony forming assays, respectively.

Results

Our current studies reveal an encompassing model for the role of RLIP76 in regulating the levels of fundamental proteins like PI3K, Akt, E-cadherin, CDK4, Bcl2 and PCNA which are of specific importance in the signal transduction from critical upstream signaling cascades that determine the proliferation, apoptosis and differentiation of pancreatic cancer cells. RLIP76 depletion also caused marked and sustained regression of established human BxPC-3 pancreatic cancer tumors in nude mouse xenograft model. RLIP76 turned out to be a major regulator of drug transport along with contributing to the radiation resistance in pancreatic cancer.

Conclusions/Significance

RLIP76 represents a mechanistically significant target for developing effective interventions in aggressive and refractory pancreatic cancers.     

MicroRNA alterations and associated aberrant DNA methylation patterns across multiple sample types in oral squamous cell carcinoma

Background

MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.

Methods

TaqMan® qRT-PCR arrays and individual assays were used to profile miRNA expression in a panel of 25 tumors with matched adjacent tissues from patients with OSCC, and 8 control paired oral stroma and epithelium from healthy volunteers. Associated DNA methylation changes of candidate epigenetically deregulated miRNA genes were measured in the same samples using the MassArray® mass spectrometry platform. MiRNA expression and DNA methylation changes were also investigated in FACS sorted CD44^high oral cancer stem cells from primary tumor samples (CSCs), and in oral rinse and saliva from 15 OSCC patients and 7 healthy volunteers.

Results

MiRNA expression patterns were consistent in healthy oral epithelium and stroma, but broadly altered in both tumor and adjacent tissue from OSCC patients. MiR-375 is repressed and miR-127 activated in OSCC, and we confirm previous reports of miR-137 hypermethylation in oral cancer. The miR-200 s/miR-205 were epigenetically activated in tumors vs normal tissues, but repressed in the absence of DNA hypermethylation specifically in CD44^high oral CSCs. Aberrant miR-375 and miR-200a expression and miR-200c-141 methylation could be detected in and distinguish OSCC patient oral rinse and saliva from healthy volunteers, suggesting a potential clinical application for OSCC specific miRNA signatures in oral fluids.

Conclusions

MiRNA expression and DNA methylation changes are a common event in OSCC, and we suggest miR-375, miR-127, miR-137, the miR-200 family and miR-205 as promising candidates for future investigations. Although overall activated in OSCC, miR-200/miR-205 suppression in oral CSCs indicate that cell specific silencing of these miRNAs may drive tumor expansion and progression.     

Flavokawain A Induces Apoptosis in MCF-7 and MDA-MB231 and Inhibits the Metastatic Process In Vitro

Introduction

The kava-kava plant (Piper methsyticum) is traditionally known as the pacific elixir by the pacific islanders for its role in a wide range of biological activities. The extract of the roots of this plant contains a variety of interesting molecules including Flavokawain A and this molecule is known to have anti-cancer properties. Breast cancer is still one of the leading diagnosed cancers in women today. The metastatic process is also very pertinent in the progression of tumorigenesis.

Methods

MCF-7 and MDA-MB231 cells were treated with several concentrations of FKA. The apoptotic analysis was done through the MTT assay, BrdU assay, Annexin V analysis, cell cycle analysis, JC-1 mitochondrial dye, AO/PI dual staining, caspase 8/9 fluorometric assay, quantitative real time PCR and western blot. For the metastatic assays, the in vitro scratch assay, trans-well migration/invasion assay, HUVEC tube formation assay, ex vivo rat aortic ring assay, quantitative real time PCR and western blot were employed.

Results

We have investigated the effects of FKA on the apoptotic and metastatic process in two breast cancer cell lines. FKA induces apoptosis in both MCF-7 and MDA-MB231 in a dose dependent manner through the intrinsic mitochondrial pathway. Additionally, FKA selectively induces a G2/M arrest in the cell cycle machinery of MDA-MB231 and G1 arrest in MCF-7. This suggests that FKA''s anti-cancer activity is dependent on the p53 status. Moreover, FKA also halted the migration and invasion process in MDA-MB231. The similar effects can be seen in the inhibition of the angiogenesis process as well.

Conclusions

FKA managed to induce apoptosis and inhibit the metastatic process in two breast cancer cell lines, in vitro. Overall, FKA may serve as a promising candidate in the search of a new anti-cancer drug especially in halting the metastatic process but further in vivo evidence is needed.     

H2S Donor,S-Propargyl-Cysteine,Increases CSE in SGC-7901 and Cancer-Induced Mice: Evidence for a Novel Anti-Cancer Effect of Endogenous H2S?

Background

S-propargyl-cysteine (SPRC), an H₂S donor, is a structural analogue of S-allycysteine (SAC). It was investigated for its potential anti-cancer effect on SGC-7901 gastric cancer cells and the possible mechanisms that may be involved.

Methods and Findings

SPRC treatment significantly decreased cell viability, suppressed the proliferation and migration of SPRC-7901 gastric cancer cells, was pro-apoptotic as well as caused cell cycle arrest at the G₁/S phase. In an in vivo study, intra-peritoneal injection of 50 mg/kg and 100 mg/kg of SPRC significantly reduced tumor weights and tumor volumes of gastric cancer implants in nude mice, with a tumor growth inhibition rate of 40–75%. SPRC also induced a pro-apoptotic effect in cancer tissues and elevated the expressions of p53 and Bax in tumors and cells. SPRC treatment also increased protein expression of cystathione-γ-lyase (CSE) in cells and tumors, and elevated H₂S levels in cell culture media, plasma and tumoral CSE activity of gastric cancer-induced nude mice by 2, 2.3 and 1.4 fold, respectively. Most of the anti-cancer functions of SPRC on cells and tumors were significantly suppressed by PAG, an inhibitor of CSE activity.

Conclusions

Taken together, the results of our study provide insights into a novel anti-cancer effect of H₂S as well as of SPRC on gastric cancer through inducing the activity of a new target, CSE.     

Comparative Study of Nanosecond Electric Fields In Vitro and In Vivo on Hepatocellular Carcinoma Indicate Macrophage Infiltration Contribute to Tumor Ablation In Vivo

Background and Aim

Recurrence and metastasis are associated with poor prognosis in hepatocellular carcinoma even in the patients who have undergone radical resection. Therefore, effective treatment is urgently needed for improvement of patients'' survival. Previously, we reported that nanosecond pulse electric fields (nsPEFs) can ablate melanoma by induction of apoptosis and inhibition of angiogenesis. This study aims to investigate the in vivo ablation strategy by comparing the dose effect of nanosecond electric fields in vitro and in vivo on hepatocellular carcinoma.

Materials and Methods

Four hepatocellular carcinoma cell lines HepG2, SMMC7721, Hep1-6, and HCCLM3 were pulsed to test the anti-proliferation and anti-migration ability of 100 ns nsPEFs in vitro. The animal model of human subdermal xenograft HCCLM3 cells into BALB/c nude mouse was used to test the anti-tumor growth and macrophage infiltration in vivo.

Results

In vitro assays showed anti-tumor effect of nsPEFs is dose-dependant. But the in vivo study showed the strategy of low dose and multiple treatments is superior to high dose single treatment. The macrophages infiltration significantly increased in the tumors which were treated by multiple low dose nsPEFs.

Conclusion

The low dose multiple nsPEFs application is more efficient than high dose single treatment in inhibiting the tumor volume in vivo, which is quite different from the dose-effect relationship in vitro. Beside the electric field strength, the macrophage involvement must be considered to account for effect variability and toxicology in vivo.     

NSC23925, Identified in a High-Throughput Cell-Based Screen,Reverses Multidrug Resistance

Background

Multidrug resistance (MDR) is a major factor which contributes to the failure of cancer chemotherapy, and numerous efforts have been attempted to overcome MDR. To date, none of these attempts have yielded a tolerable and effective therapy to reverse MDR; thus, identification of new agents would be useful both clinically and scientifically.

Methodology/Principal Findings

To identify small molecule compounds that can reverse chemoresistance, we developed a 96-well plate high-throughput cell-based screening assay in a paclitaxel resistant ovarian cancer cell line. Coincubating cells with a sublethal concentration of paclitaxel in combination with each of 2,000 small molecule compounds from the National Cancer Institute Diversity Set Library, we identified a previously uncharacterized molecule, NSC23925, that inhibits Pgp1 and reverses MDR1 (Pgp1) but does not inhibit MRP or BCRP-mediated MDR. The cytotoxic activity of NSC23925 was further evaluated using a panel of cancer cell lines expressing Pgp1, MRP, and BCRP. We found that at a concentration of >10 µM NSC23925 moderately inhibits the proliferation of both sensitive and resistant cell lines with almost equal activity, but its inhibitory effect was not altered by co-incubation with the Pgp1 inhibitor, verapamil, suggesting that NSC23925 itself is not a substrate of Pgp1. Additionally, NSC23925 increases the intracellular accumulation of Pgp1 substrates: calcein AM, Rhodamine-123, paclitaxel, mitoxantrone, and doxorubicin. Interestingly, we further observed that, although NSC23925 directly inhibits the function of Pgp1 in a dose-dependent manner without altering the total expression level of Pgp1, NSC23925 actually stimulates ATPase activity of Pgp, a phenomenon seen in other Pgp inhibitors.

Conclusions/Significance

The ability of NSC23925 to restore sensitivity to the cytotoxic effects of chemotherapy or to prevent resistance could significantly benefit cancer patients.     

Telomere Shortening Sensitizes Cancer Cells to Selected Cytotoxic Agents: In Vitro and In Vivo Studies and Putative Mechanisms

Background

Telomere/telomerase system has been recently recognized as an attractive target for anticancer therapy. Telomerase inhibition results in tumor regression and increased sensitivity to various cytotoxic drugs. However, it has not been fully established yet whether the mediator of these effects is telomerase inhibition per se or telomere shortening resulting from inhibition of telomerase activity. In addition, the characteristics and mechanisms of sensitization to cytotoxic drugs caused by telomerase inhibition has not been elucidated in a systematic manner.

Methodology/Principal Findings

In this study we characterized the relative importance of telomerase inhibition versus telomere shortening in cancer cells. Sensitization of cancer cells to cytotoxic drugs was achieved by telomere shortening in a length dependent manner and not by telomerase inhibition per se. In our system this sensitization was related to the mechanism of action of the cytotoxic drug. In addition, telomere shortening affected also other cancer cell functions such as migration. Telomere shortening induced DNA damage whose repair was impaired after administration of cisplatinum while doxorubicin or vincristine did not affect the DNA repair. These findings were verified also in in vivo mouse model. The putative explanation underlying the phenotype induced by telomere shortening may be related to changes in expression of various microRNAs triggered by telomere shortening.

Conclusions/Significance

To our best knowledge this is the first study characterizing the relative impact of telomerase inhibition and telomere shortening on several aspects of cancer cell phenotype, especially related to sensitivity to cytotoxic drugs and its putative mechanisms. The microRNA changes in cancer cells upon telomere shortening are novel information. These findings may facilitate the development of telomere based approaches in treatment of cancer.     

Interaction between Alcohol Consumption and CYP 2C19 Gene Polymorphism in Relation to Oesophageal Squamous Cell Carcinoma

Objectives

The purpose of this study is to explore the relationship between the interactions of CYP2C19 gene polymorphisms and several environmental factors and oesophageal squamous cell carcinoma (OSCC).

Methods

In a case-control study of OSCC patients (n = 350) and healthy controls (n = 350), we investigated the roles of polymorphism in the CYP2C19 gene by the use of polymerase chain reaction - restriction fragment length polymorphism (PCR – RFLP) analysis.

Results

The CYP2C19^*3 AG+AA genotype was significantly more prevalent in OSCC patients (10.0% versus 3.43%; P<0.01). Multiple logistic regression analysis showed drinking (OR: 5.603, 95% CI: 3.431–11.112; P = 0.005) and smoking (OR: 4.341, 95% CI: 3.425–10.241; P = 0.001) was the independent risk factor of OSCC respectively, and there were significant interaction between CYP2C19^*3 and drinking (OR: 8.747, 95% CI: 6.321–18.122; P = 0.009).

Conclusions

The CYP2C19^*3 polymorphism and OSCC were synergistically and significantly associated in Chinese Han patients.     

USP22 Is Useful as a Novel Molecular Marker for Predicting Disease Progression and Patient Prognosis of Oral Squamous Cell Carcinoma

Background and Objective

The significance of ubiquitin-specific protease 22 (USP22) as a potential marker has been growing in the field of oncology. The aim of this study was to investigate the role of USP22 and the association with its potential targets in oral squamous cell carcinoma (OSCC).

Methods

Immunohistochemistry was used to determine the expression of USP22 protein in 319 OSCC patients in comparison with 42 healthy controls. The clinical correlations and prognostic significance of the aberrantly expressed protein was evaluated to identify novel biomarker of OSCC.

Results

The incidence of positive USP22 expression was 63.32% in 319 conventional OSCC tissues. The protein expression level of USP22 was concomitantly up-regulated from non-cancerous mucosa to primary carcinoma and from carcinomas to lymph node metastasis (P<0.001). Moreover, statistical analysis showed that positive USP22 expression was positively related to lymph node metastasis, Ki67, Cox-2 and recurrence. Furthermore, it was shown that patients with positive USP22 expression had significantly poorer outcome compared with patients with negative expression of USP22 for patients with positive lymph nodes. Multivariate Cox regression analysis revealed that USP22 expression level was an independent prognostic factor for both overall survival and disease-free survival (P<0.001 and P<0.001, respectively). Cancer cells with reduced USP22 expression exhibited reduced proliferation and colony formation evaluated by MTT and soft agar assays.

Conclusion

To our knowledge, this is the first study that determines the relationship between USP22 expression and prognosis in OSCC. We found that increased expression of USP22 is associated with poor prognosis in OSCC. USP22 may represent a novel and useful prognostic marker for OSCC.     

Antihelminthic benzimidazoles potentiate navitoclax (ABT-263) activity by inducing Noxa-dependent apoptosis in non-small cell lung cancer (NSCLC) cell lines

Background

Evasion of apoptosis is a hallmark of cancer cells. One mechanism to deregulate the apoptotic pathway is by upregulation of the anti-apoptotic Bcl-2 family members. Navitoclax (ABT-263) is a Bcl-2/Bcl-x_L inhibitor that restores the ability of cancer cells to undergo apoptosis.

Methods

In this study we performed a high-throughput screen with 640 FDA-approved drugs to identify potential therapeutic combinations with navitoclax in a non-small cell lung cancer (NSCLC) cell line.

Results

Other than a panel of cancer compounds such as doxorubicin, camptothecin, and docetaxel, four antihelminthic compounds (benzimidazoles) potentiated navitoclax activity. Treatment with benzimidazoles led to induction of the pro-apoptotic protein Noxa at the mRNA and protein level. Noxa binds and antagonizes antiapoptotic protein Mcl-1. siRNA-mediated knock-down of Noxa completely rescued benzimidazole-potentiated navitoclax activity. In addition, inhibiting caspase 3 and 9 partially rescued benzimidazole-potentiated navitoclax activity.

Conclusions

We have identified compounds and mechanisms which potentiate navitoclax activity in lung cancer cell lines. Further validation of the benzimidazole-potentiated navitoclax effect in vivo is required to evaluate the potential for translating this observation into clinical benefit.

Electronic supplementary material

The online version of this article (doi:10.1186/s12935-014-0151-3) contains supplementary material, which is available to authorized users.