Synergistic Effect Of Doxorubicin Research Articles

Synergistic Effect of Doxorubicin and Blue Light Irradiation on the Antitumor Treatment of HepG2 Cells in Liver Cancer.

Doxorubicin (DOX) has been an effective antitumor agent for human liver cancer cells; however, an overdose might lead to major side effects appearing in clinical applications. In this work, we present a strategy of combining DOX and blue light (BL) irradiation for the antitumor treatment of HepG2 cells (one typical human liver cancer cell line). It is demonstrated that synergetic DOX and BL can significantly reduce cell proliferation and increase the apoptotic rate of HepG2 cells in comparison to individual DOX treatment. The additional BL irradiation is further helpful for enhancing the inhibition of cell migration and invasion. Analyses of reactive oxygen species (ROS) level and Western blotting reveal that the strategy results in more ROS accumulation, mitochondrial damage, and the upregulation of proapoptotic protein (Bcl-2) and downregulation of antiapoptotic protein (Bax). In addition to the improved therapeutic effect, the non-contact BL irradiation is greatly helpful for reducing the dosage of DOX, and subsequently reduces the side effects caused by the DOX drug. These findings offer a novel perspective for the therapeutic approach toward liver cancer with high efficiency and reduced side effects.

Cellular immunity analysis by a modular acoustofluidic platform: CIAMAP.

The study of molecular mechanisms at the single-cell level holds immense potential for enhancing immunotherapy and understanding neuroinflammation and neurodegenerative diseases by identifying previously concealed pathways within a diverse range of paired cells. However, existing single-cell pairing platforms have limitations in low pairing efficiency, complex manual operation procedures, and single-use functionality. Here, we report a multiparametric cellular immunity analysis by a modular acoustofluidic platform: CIAMAP. This platform enables users to efficiently sort and collect effector-target (i.e., NK92-K562) cell pairs and monitor the real-time dynamics of immunological response formation. Furthermore, we conducted transcriptional and protein expression analyses to evaluate the pathways that mediate effector cytotoxicity toward target cells, as well as the synergistic effect of doxorubicin on the cellular immune response. Our CIAMAP can provide promising building blocks for high-throughput quantitative single-cell level coculture to understand intercellular communication while also empowering immunotherapy by precision analysis of immunological synapses.

Transdermal delivery of doxorubicin and methotrexate from polyelectrolyte three layer nanoparticle of graphene oxide/polyethyleneimine/dextran sulphate for chemotherapy: In vitro and in vivo studies

In the present work, novel dual drug loaded - anionic graphene oxide/cationic polyethyleneimine/poly anionic dextran sulfate (GO/PEI/DS) was synthesized by a layer-by-layer self-assembly technique for transdermal anti-cancer drug delivery. Doxorubicin (DOX) was loaded onto folic acid conjugated graphene oxide (GO) and methotrexate (MTX) was loaded onto dextran sulfate (DS). The drug carrier was characterized by fourier transform infrared spectroscopy, x-ray diffraction spectroscopy, dynamic light scattering analysis, scanning electron microscopy, atomic force microscopy, transmission electron microscopy, zeta-potential analysis, drug loading and encapsulation efficiency, equilibrium swelling studies, in vitro drug release studies, hemocompatibility assays, in vitro cytotoxicity assay, skin irritation studies, ex vivo skin permeation studies and in vivo pharmacokinetic studies.The cytotoxicity of synthesized materials was evaluated against MCF-7 breast cancer cells using an MTT assay and apoptosis assay. The synergistic effect of DOX and MTX to inhibit the growth of cancer cells was also confirmed. The final dual drug-loaded carrier showed a cell viability of 36.38 % towards MCF-7 cancer cells while it showed a cell viability of >87.65 % towards normal L929 cell lines. The in vivo pharmacokinetic studies in Wistar rats through oral and transdermal routes of administration, the transdermal route of administration of dual drug-loaded material provided a sustained and prolonged drug existence in the blood circulation system. The mean residence time of drugs in the bloodstream via oral administration was 83.98 ± 3.71 h, while that of the transdermal route of administration was 149.62 ± 6.11 h. The results revealed that the synthesized dual drug-loaded material showed a good pH-dependent controlled and sustained release profile for both DOX and MTX via the transdermal route of administration in comparison with oral delivery.

Search for immunomodulatory compounds with antiproliferative activity against melanoma

BackgroundMelanoma is a highly aggressive neoplasm with a high degree of malignancy and rapid acquisition of resistance by cancer cells. MethodsBiological studies of a series of isoxazole compounds with immunomodulatory properties were preceded by in silico analysis. The assay evaluated the viability of NHDF and A375 cell cultures after the administration of isoxazole compounds after a 24-hour incubation period in the MTT test. Analyzes of ROS and NO scavenging, P-glycoprotein activity, and properties were performed. The levels of Caspase 3 and Caspase 9 were measured using ELISA to assess which pathways induced apoptosis by the tested compounds. On the chip, the synergistic effect of doxorubicin and the most active compound from the MM9 series on cells of the A375 melanoma line was determined. ResultsAll tested N′-substituted derivatives of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide with immunomodulatory activity show multidirectional antitumor activity on A375 melanoma lines with an affinity for P-glycoprotein, induction of free radical formation and generation of DNA damage leading to the death of cancer cells, as well as formation of complexes with DNA Topoisomerase II. Most of the tested compounds show pro-apoptotic activity. The most active compound in the series induces apoptosis in three distinct pathways and acts synergistically with doxorubicin. ConclusionsThe most active compound with immunomodulatory properties showed multidirectional antitumor activity against cells of the A375 melanoma line and also had a synergistic pro-apoptotic effect with doxorubicin, which may result in a reduction of this cytostatic dose with increased effectiveness.

Abstract 2441: hetIL-15 decreases tumor cell dissemination and colonization and synergizes with chemotherapy and surgery to cure murine 4T1 breast tumors

Abstract Introduction: Metastasis is responsible for most of the cancer-related deaths (~ 90%). Metastatic triple negative breast cancer (TNBC) has poor survival and chemotherapy pre- and post-surgery has been the standard treatment for decades. Polymorphonuclear - myeloid derived suppressor cells (PMN-MDSCs) are implicated in the process of metastasis. IL-15 stimulates the proliferation and cytolytic activity of CD8+T cells and Natural Killer cells (NKs) and has been reported to have anti-metastatic activity. We have produced the native heterodimeric form of IL-15 (hetIL-15) which has advanced in clinical trials due to its anticancer activities. Here, we report the anti-metastatic effects of hetIL-15 alone and in combination with doxorubicin and surgery using the 4T1 mouse model of TNBC. Study design and methods: We studied the efficacy of the chemo-immunotherapeutic regimen with surgery in a neoadjuvant and adjuvant setting or without surgery. The animals were evaluated regarding metastatic disease and survival. The anti-metastatic effect was evaluated by examining the metastatic foci in lungs and the circulating tumor cells (CTCs) in blood by histology and clonogenic assays. Treatment effects in immune populations in blood, spleen and lungs were evaluated by flow cytometry analysis and immunohistochemistry (IHC). Results: Mice treated with hetIL-15 in combination with doxorubicin had significantly better survival in comparison to monotherapy treatments and control group. H&E histological analysis in the lungs revealed substantially fewer metastatic foci after hetIL-15 monotherapy, and even fewer in the combination group. Furthermore, clonogenic assays from lungs and blood, reinforced these findings showing significantly lower numbers of tumor colonies upon hetIL-15 monotherapy and combination treatment. Immune profiling evaluation of blood, spleen and lungs by flow cytometry and IHC revealed a significant systemic increase of cytotoxic effector cells (CD8+T cells and NKs), combined with reduction of immunosuppressive populations (PMN-MDSCs) especially in the combination group. These findings suggest a synergistic effect of doxorubicin and hetIL-15 in controlling the metastatic disease. Lastly, hetIL-15 monotherapy or co-administration with doxorubicin, together with surgery, led to the cure of approximately 50% of the treated mice. Conclusions: Our results demonstrate that hetIL-15 reduces metastatic disease and synergizes with doxorubicin and surgery to maximize the effectiveness of the treatment against breast cancer in mice. We suggest that the effect of hetIL-15 on metastatic disease is both at the level of dissemination and also colonization in the metastatic sites. This should be further explored in the clinical setting as a neoadjuvant and adjuvant therapy in combination with chemotherapy and surgery for the treatment of TNBC. Citation Format: Vasiliki Stravokefalou, Dimitris Stellas, Sevasti Karaliota, Breana Myers, Cristina Bergamaschi, Konstantinos Dimas, Barbara K. Felber, George N. Pavlakis. hetIL-15 decreases tumor cell dissemination and colonization and synergizes with chemotherapy and surgery to cure murine 4T1 breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2441.

Synergistic Effect of Doxorubicin and siRNA-Mediated Silencing of Mcl-1 Using Cationic Niosomes against 3D MCF-7 Spheroids.

Chemotherapy is a vital option for cancer treatment; however, its therapeutic outcomes are limited by dose-dependent toxicity and the occurrence of chemoresistance. siRNAs have emerged as an attractive therapeutic option enabling specific interference with target genes. Combination therapy using chemotherapeutic agents along with gene therapy could be a potential strategy for cancer management, which not only improves therapeutic efficacy but also decreases untoward effects from dose reduction. In this study, a cationic niosome containing plier-like cationic lipid B was used to convey siRNA against anti-apoptotic mRNA into MCF-7 and MDA-MB-231 cells. Mcl-1 silencing markedly decreased the viability of MCF-7 cells and triggered apoptosis. Moreover, computer modeling suggested that the combination of doxorubicin (Dox) and Mcl-1 siRNA exhibited a synergistic relationship and enabled a dose reduction of each agent at 1.71 and 3.91 folds, respectively, to reach a 90% inhibitory effect when compared to single-agent treatments. Synergistic antitumor activity was further verified in a 3D spheroid culture which revealed, in contrast to single-agent treatment, the combination markedly decreased spheroid volume over time. Together, the combination therapy between Mcl-1 silencing and Dox exhibits a synergistic effect that may be exploited for novel breast cancer treatment.

PH-sensitive micelles self-assembled from star-shaped TPGS copolymers with ortho ester linkages for enhanced MDR reversal and chemotherapy

TPGS approved by FDA can be used as a P-gp inhibitor to effectively reverse multi-drug resistance (MDR) and as an anticancer agent for synergistic antitumor effects. However, the comparatively high critical micelle concentration (CMC), low drug loading (DL) and poor tumor target limit its further clinical application. To overcome these drawbacks, the pH-sensitive star-shaped TPGS copolymers were successfully constructed via using pentaerythritol as the initial materials, ortho esters as the pH-triggered linkages and TPGS active-ester as the terminated MDR material. The amphiphilic star-shaped TPGS copolymers could self-assemble into free and doxorubicin (DOX)-loaded micelles at neutral aqueous solutions. The micelles exhibited the lower CMC (8.2 × 10−5 mg/ml), higher DL (10.8%) and long-term storage and circulation stability, and showed enhanced cellular uptake, apoptosis, cytotoxicity, and growth inhibition for in vitro MCF-7/ADR and/or MCF-7/ADR multicellular spheroids and in vivo MCF-7/ADR tumors via efficiently targeted drug release at tumoral intracellular pH (5.0), MDR reversal of TPGS, and synergistic effect of DOX and TPGS. Therefore, the pH-sensitive micelles self-assembled from star-shaped TPGS copolymers with ortho ester linkages are potentially useful to clinically transform for enhanced MDR cancer treatment.

Selection of Effective Therapies Using Three-Dimensional in vitro Modeling of Chondrosarcoma.

Purpose: Chondrosarcomas are a group of cartilaginous malignant neoplasms characterized by the deposition of chondrogenic extracellular matrix. Surgical resection is currently the only curative treatment option, due to their high resistance to conventional chemotherapy and radiotherapy. Novel therapeutic treatment options may improve outcome. Predominantly used cell line monolayer in vitro models lack in vivo complexity, such as the presence of extracellular matrix, and differing oxygen access. Hence, we aimed to improve pre-clinical chondrosarcoma research by developing an alginate-based 3D cell culture model.Method: An alginate scaffold was applied to generate spheroids of three chondrosarcoma cell lines (CH2879, JJ012, SW1353). Morphological, histological and immunohistochemical assessment of the spheroids were used to characterize the chondrosarcoma model. Presto blue assay, morphological and immunohistochemical assessment were applied to assess spheroid response to a panel of chemotherapeutics and targeted therapies, which was compared to conventional 2D monolayer models. Synergistic effect of doxorubicin and ABT-737 (Bcl-2 inhibitor) was compared between monolayer and spheroid models using excess over Bliss. A 3D colony formation assay was developed for assessment of radiotherapy response.Results: Chondrosarcoma spheroids produced chondrogenic matrix and remained proliferative after 2 weeks of culture. When treated with chemotherapeutics, the spheroids were more resistant than their monolayer counterparts, in line with animal models and clinical data. Moreover, for sapanisertib (mTOR inhibitor) treatment, a recovery in chondrosarcoma growth, previously observed in mice models, was also observed using long-term treatment. Morphological assessment was useful in the case of YM-155 (survivin inhibitor) treatment where a fraction of the spheroids underwent cell death, however a large fraction remained proliferative and unaffected. Synergy was less pronounced in 3D compared to 2D. A 3D clonogenic assay confirmed increased resistance to radiotherapy in 3D chondrosarcoma spheroids.Conclusion: We demonstrate that the chondrosarcoma alginate spheroid model is more representative of chondrosarcoma in vivo and should be used instead of the monolayer model for therapy testing. Improved selection at in vitro stage of therapeutic testing will increase the amount of information available for experimental design of in vivo animal testing and later, clinical stages. This can potentially lead to increased likelihood of approval and success at clinical trials.

Trifolium pratense L. (red clover) extract and doxorubicin synergistically inhibits proliferation of 4T1 breast cancer in tumor-bearing BALB/c mice through modulation of apoptosis and increase antioxidant and anti-inflammatory related pathways.

Therapeutic strategies against triple‐negative breast cancer (TNBC) are associated with drug‐induced toxicities. The tropical edible red clover (Trifolium pratense L.) is rich in polyphenolic compounds which confer the plant potential anticancer properties. The aim of this study was to investigate the effects of T. pratense and doxorubicin (DOX) on the apoptosis and proliferation of 4T1 tumor cells in an allograft model of tumor‐bearing BALB/c mice. Fifty‐six female 4T1‐tumor bearing‐ BALB/c mice were randomly divided into 7 groups (n = 8/group) to receive different doses and combinations of DOX and T. pratense extract for 35 days. On the 36th day, serum estradiol (E2), IL‐12 and IFN‐γ cytokines, and glutathione peroxidase (GPx) activity were measured. Tumor's ferric reducing antioxidant power (FRAP) and the expressions of apoptosis‐related genes (p53, Bax, Bcl‐2, and caspase‐3) were also evaluated. Immunohistochemical staining for Ki‐67 and p53 were performed. Our results showed that the co‐treatment of DOX and T. pratense (100–400 mg/kg) inhibited the proliferation of 4T1 tumor cells in dose‐ and time‐dependent manners. The co‐treatment of DOX and T. pratense (especially at the dose of 400 mg/kg) decreased the serum level of E2 (as a stimulant for breast tumor growth) and increased the serum levels of IL‐12 and IFN‐γ along with significant increments in serum GPx and tumor FRAP activities. The co‐administration of DOX and T. pratense also decreased the expression of Ki‐67 proliferation marker and increased the number p53 positive (i.e., apoptotic) cells within tumors. This was accompanied with the upregulation of pro‐apoptotic and down‐regulation of antiapoptotic genes. The key findings indicated the synergistic effects of DOX and T. pratense against TNBC xenografts.

Nanoparticle mediated codelivery of nifuratel and doxorubicin for synergistic anticancer therapy through STAT3 inhibition

Chemotherapy is one of the most potent strategies to treat gastric cancer in clinic. However, the resistance of cancer cells to chemotherapeutics is a remarkable impediment to the treatment. Moreover, signal transducer and activator of transcription 3 (STAT3) is a critical transcriptional factor that over-activated in gastric cancer, and highly involved in the induction of chemoresistance. In this study, we developed poly (lactic-co-glycolic acid) (PLGA) nanoparticles to achieve the simultaneous codelivery of doxorubicin (DOX) and nifuratel (NIF, a novel STAT3 inhibitor) for enhanced cancer therapy. The synergistic effect of DOX and NIF against cancer cells was evaluated in gastric cancer cells. PLGA nanoparticles with an optimal ratio of DOX and NIF (DNNPs) were prepared and characterized. The cellular uptake and anticancer effects of DNNPs were investigated, and the underlying mechanisms were further explored. DNNPs presented as a spherical shape, provided sustained release profiles, and exhibited significantly increased uptake and cytotoxicity in gastric cancer cells. Mechanism studies showed that DNNPs significantly induced mitochondrial-dependent apoptosis and inhibited STAT3 phosphorylation, explaining the enhanced anticancer effect. These results suggested that DNNPs represented a promising strategy against gastric cancer by inhibiting the STAT3 pathway and amplifying apoptosis.

Abstract 4901: Inhibition of γ-secretase sensitizes drug-resistant neuroblastoma cells to doxorubicin

Abstract Introduction: Neuroblastoma is the most common extracranial solid tumor in childhood and accounts for ~15% of all pediatric cancer mortality. Approximately 650 new cases are reported annually in the USA, with 90% of cases diagnosed by 5 years of age. Novel therapies are needed as ~50% of cases are high-risk neuroblastoma with a poor (<50%) five-year survival rate due to relapse and treatment resistance. Neuroblastoma cells undergo a mesenchymal change, including the activation of Notch pathway genes, in their transition to a drug-resistant state. The Notch pathway has both oncogenic and tumor-suppressive roles and has been implicated in epithelial-mesenchymal transition (EMT) and chemoresistance. This pathway can be blocked by γ-secretase inhibition, which prevents cleavage of the Notch receptor and therefore Notch signal transduction. Thus, targeting the Notch pathway has been considered a promising anticancer strategy. Underlying mechanisms of the Notch signaling pathway in drug-resistant cancers, including neuroblastoma, are not well understood. Aim: Determine if inhibition of the Notch signaling pathway sensitizes chemoresistant neuroblastoma cells to doxorubicin. Methods: Doxorubicin-resistant (DoxR) cells were generated by incubating doxorubicin-sensitive, wild-type (WT) patient-derived cell lines with incremental doxorubicin concentrations (1 x 10-9 to 1 x 10-6 M) over six months. Cells were considered chemoresistant after surviving five consecutive passages in 1 µM doxorubicin (MTT assay). Differentially expressed genes were identified by microarray (Illumina HumanHT-12 v4 Expression BeadChip) and validated by RT-qPCR. Cells treated with doxorubicin and/or DAPT (γ-secretase inhibitor) were evaluated for cell viability (MTT assay), proliferation (PCNA immunofluorescence and Western blot), senescence (SA-β-Gal immunocytochemistry and p21 Western blot) and proteomic profiling (2D-DIGE). Results: Compared to WT cells, DoxR cells differentially expressed the following Notch pathway genes: downregulation of DLK1 and upregulation of HEY1 and SNAI2, as determined by microarray and RT-qPCR. Compared to WT cells, DoxR cells treated with both doxorubicin and DAPT exhibited significantly decreased cell viability, decreased proliferation and increased senescence. DAPT-treated DoxR cells differentially expressed nine proteins using 2D-DIGE: MCM3, MCM6, CAPN10, ACTN4, TUBA1B, HNRNPK, PDP1, NPM1 and BARD1. Conclusion: The γ-secretase inhibitor DAPT sensitizes DoxR neuroblastoma cells to doxorubicin. Mechanisms underlying the synergistic effect of doxorubicin and DAPT in DoxR cells warrant further investigation. Citation Format: Kathryn A. Solka, Fei Chu, Yi-Yong Qiu, Sandra H. Clark, Mary Beth Madonna. Inhibition of γ-secretase sensitizes drug-resistant neuroblastoma cells to doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4901.

Liposomal Codelivery of Doxorubicin and Andrographolide Inhibits Breast Cancer Growth and Metastasis.

Effective treatment of metastatic (stage IV) breast cancers remains a formidable challenge. To address this issue, a cell-penetrating peptide-assisted liposomal system was developed for codelivery of doxorubicin and andrographolide. This nanomedicine-based combination therapy showed the ability to inhibit the in vitro migration and invasion of 4T1 cells through the wound healing and transwell invasion assays. Furthermore, this delivery system exhibited the enhanced accumulation in the tumor tissues and deep intratumoral penetration. The synergistic effect of doxorubicin and andrographolide led to an evident inhibition of tumor growth in an orthotopic breast tumor mouse model and efficient prevention of lung metastasis. The therapeutic mechanism was associated with the anti-angiogenesis effect. In conclusion, this nanomedicine-based combination therapy provides a potential method for overcoming metastatic breast cancers.

Synergistic Effects of Doxorubicin and Cardenolid Glycosides of Calotropis Gigantea Root on Cervical Cancer Hela Cell Line

Cardenolid glycosides (CGs) has been known has high anticancer activity against several types of cancers, for example, breast cancer, lung cancer, prostate cancer, melanoma, neuroblastoma, myeloma and leukemia in the in vitro and in vivo research. The aim of our study was to know the synergistic potency of CGs improving the efficacy of doxorubicin in cervical cancer. Activities from combination of doxorubicin and CGs was measured by MTT colorimetric methode. Combination Index was used as combination efficacy parameter. The results showed that the IC50 of CGs was 1,023 μg/mL. CGs demonstrated selective activity in inhibiting the growth of HeLa cells by selectivity index > 3 (241.9). Combination test for CGs and Doxorubicin showed strong synergistic effect in doses 200 ug/mL Doxorubicin and 1.79 ug/mL CGs. The synergistic effect has been shown by 10 combination doses for the doses below the IC50. CGs increases the efficacy of Doxorubicin for the doses below the IC50, thus CGs can be recommended as co chemotherapy in cervical cancer treatment.

The Synergistic Effect of Doxorubicin and Ethanolic Extracts of Caesalpinia sappan L. Wood and Ficus septica Burm. f. Leaves on Viability, Cell Cycle Progression, and Apoptosis Induction of MCF­7 Cells

Caesalpinia sappan L. and Ficus septica Burm. f known asa potential plant with wide variety of medicinal properties, including anticancer. Present study was aimed to explore cytotoxic effect ofsappan wood (ECS) and awar-awar leaves (EFS), and its combination with doxorubicin (dox) on MCF-7 cells focusing on cell cycle progression and apoptosis induction.The result of MTT assay showed that single treatment of ECS and dox performed cytotoxic effect with the IC50 value of 32 µg/mL and 6 µM respectively, while EFS performed low cytotoxic effect with the IC50 value of 282 µg/mL. The combination of ECS with EFS and doxorubicin showed synergistic cytotoxic effect. Flow cytometry analysis revealed that combination of ECS (16 µg/mL) with EFS (8 µg/mL) and doxorubicin (2 µM) induced apoptosis, and cell accumulation at sub-G1 and G2/M phases.Immunoblotting assay confirmed the apoptosis induction of this combination through increasing of cleavage of PARP-1. Based on these results, the synergistic cytotoxic effect of this combinationwas through G2/M phase accumulation and apoptosis inductionand potentially to be developed as co-chemotherapeutic agent.

672. Hybrid AAV/Phage Vector Enhances Chemotherapy Efficacy Against Cancer

Chemotherapy has been widely used for cancer treatment, both in early and late stages. However, chemotherapy does not selectively target tumor cells, as normal cells are also harmed by the drugs resulting in several side effects. Another major obstacle to the success of chemotherapy in cancer treatment is the development of tumor drug resistance by cancer cells. In order to avoid these problems, the combination of chemotherapy with other therapeutic strategies has been used in order to lower the chemotherapeutic drug dose. Gene therapy is one of the therapeutic strategies that can be combined with chemotherapy. Mammalian viruses are well recognized vehicles for gene therapy, but major drawbacks of these viral vectors are broad tissue tropism following systemic administration (low specificity for the target cells/tissues), their fragility to harsh environments and difficulty for large scale production. Therefore, our group has developed a vector from bacteriophage, a bacteria virus also named phage. This novel engineered phage, called AAV/Phage, displays the RGD4C peptide to target a specific receptor (αvβ3 integrin) on cancer cell surface, while the phage genome is merged with recombinant rAAV2 virus genome carrying the transgene to deliver. In comparison with mammalian viral vectors, the production of AAV/Phage is quicker, simpler and more economical. In addition, the vector is stable at 4oC for many years. The AAV/Phage vector efficiently targeted, delivered, and expressed transgene in cancer cells in vitro. We also proved that the vector selectively targeted gene delivery to tumors after intravenous injection in animal models of cancer. Moreover, in brain tumor models, the vector can penetrate through blood brain barrier and selectively delivers transgene expression to brain tumors. We used our vector as combination therapies with some well-known cancer drugs, such as doxorubicin, and temozolomide. Combination treatment of AAV/Phage vector carrying the Herpes Simplex virus thymidine kinase gene (AAV/Phage-HSVtk.) with doxorubicin increased the targeted cancer cell killing in 2D tissue cultures and 3D tumor spheroids of rat gliosarcoma (9L) and human melanoma (M21) cells. We found that this increase in tumor cell killing was associated with a synergistic effect of doxorubicin on enhancing gene expression by AAV/Phage. We then combined AAV/Phage carrying short hairpin RNA to suppress mTOR gene expression (AAV/Phage-shmTOR) with temozolomide to treat medulloblastoma cells (DAOY). The results exhibited that treatment of medulloblastma with the vector alone efficiently suppresses the expression of mTOR gene, but has no effect on cell killing. Treatment of temozolomide at low dose (500 uM) did not have effect on cell killing, but combination therapies of temozolomide with AAV/Phage-shmTOR significantly increased cell death. Altogether, our results demonstrate that combination of AAV/Phage carrying therapeutic genes with cancer chemotherapeutic drugs is an effective strategy for cancer treatment. In future work, we plan to investigate the efficacy of AAV/Phage and cancer drug combination treatment in pre-clinical models of cancer.

Synergistic growth inhibitory effect of deracoxib with doxorubicin against a canine mammary tumor cell line, CMT-U27.

Cyclooxygenase (COX) inhibitors have been shown to exert anti-angiogenic and anti-tumor activities on many types of malignant tumors. These anticancer properties make it worthwhile to examine the possible benefit of combining COX inhibitors with other anti-cancer agents. In the present study, we evaluated the potential of deracoxib (DER) in potentiating antitumor activity of doxorubicin (DOX) in canine mammary carcinoma cells (CMT-U27). DER (50–250 µM) enhanced the antiproliferative activity of DOX by reducing the IC50 (approximately 3- to 3.5 fold). Interaction analysis of the data showed that combinations of DOX at 0.9 µM with DER (100–250 µM) produced synergism in the CMT-U27 cell line, with a ratio index ranging from 1.98 to 2.33. In additional studies identifying the mechanism of observed synergistic effect, we found that DER strongly potentiated DOX-caused G0/G1 arrest in cell cycle progression. Also, DER (100–250 µM) augmented apoptosis induction with approximately 1.35- and 1.37- fold increases in apoptotic response caused by DOX in the cells. DER enhanced the antiproliferative effect of DOX in conjunction with induction of apoptosis by modulation of Bcl-2 expression and changes in the cell cycle of the CMT-U27 cell line. Although the exact molecular mechanism of the alterations in the cell cycle and apoptosis observed with DER and DOX combinations require further investigations, the results suggest that the synergistic effect of DOX and DER combinations in CMT therapy may be achieved at relatively lower doses of DOX with lesser side effects. Therefore, combining DER with DOX may prove beneficial in the clinical treatment of canine mammary cancer.

A novel nanoparticle formulation overcomes multiple types of membrane efflux pumps in human breast cancer cells.

Multidrug resistance (MDR) in cancer cells can involve overexpression of different types of membrane drug efflux pumps and other drug resistance mechanisms. Hence, inhibition of one resistance mechanism may not be therapeutically effective. Previously we demonstrated a new polymer lipid hybrid nanoparticle (PLN) system was able to circumvent drug resistance of P-glycoprotein (P-gp) overexpressing breast cancer cells. The objectives of the present study were 2-fold: (1) to evaluate the ability of the PLN system to overcome two other membrane efflux pumps-multidrug resistance protein 1 (MRP1+) and breast cancer resistance protein (BCRP+) overexpressed on human breast cancer cell lines MCF7 VP (MRP1+) and MCF7 MX (BCRP+); and (2) to evaluate possible synergistic effects of doxorubicin (Dox)-mitomycin C (MMC) in these cell lines. These objectives were accomplished by measuring in vitro cellular uptake, intracellular trafficking, and cytotoxicity (using a clonogenic assay and median effect analysis), of Dox, MMC, or Dox-MMC co-loaded PLN. Treatment of MDR cells with PLN encapsulating single anticancer agents significantly enhanced cell kill compared to free Dox or MMC solutions. Dox-MMC co-loaded PLN were 20-30-folds more effective in killing MDR cells than free drugs. Co-encapsulated Dox-MMC was more effective in killing MDR cells than single agent-encapsulated PLN. Microscopic images showed perinuclear localization of fluorescently labelled PLN in all cell lines. These results are consistent with our previous results for P-gp overexpressing breast cancer cells suggesting the PLN system can overcome multiple types of membrane efflux pumps increasing the cytotoxicity of Dox-MMC at significantly lower doses than free drugs.

On the Synergistic Effect of Doxorubicin and Mitomycin C Against Breast Cancer Cells

The combination of doxorubicin and mitomycin C has been shown previously to result in supra-additive tumor cell killing in vitro in both murine and human breast cancer cells and in vivo against murine breast cancer cells. Median effect analysis was used to determine the significance and degree of interaction. The origin of this synergy was sought by evaluating the contribution of membrane efflux pump modulation, formaldehyde production, reactive oxygen species, DNA cross-linking, and DNA double-strand breaks to this effect. The interaction of mitomycin C and doxorubicin in vitro was found to be a true synergy whose mechanism was efflux pump-independent. DNA cross-links were only found to increase additively with co-administration of the drugs; however, a supra-additive increase in DNA double-strand breaks was observed. The results suggest that poisoning of topoisomerase IIalpha by doxorubicin may interact with drug-induced DNA cross-links to enhance the formation of DNA double-strand breaks. This interaction, together with glutathione depletion and mitomycin C-derived formaldehyde, may be the underlying mechanism(s) of the synergy observed between mitomycin C and doxorubicin.

Trail-induced apoptosis and interaction with cytotoxic agents in soft tissue sarcoma cell lines

Five human soft tissue sarcoma (STS) cell lines (HTB-82 rhabdomyosarcoma, HTB-91 fibrosarcoma, HTB-92 liposarcoma, HTB-93 synovial sarcoma and HTB-94 chondrosarcoma) were analysed for their sensitivity to tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and the function of the TRAIL apoptotic pathway in these cells. TRAIL induced significant apoptosis (>90%) in HTB-92 and HTB-93 cells, whereas no effect was observed in HTB-82, HTB-91 and HTB-94 cells. TRAIL-Receptor 1 (TRAIL-R1) was expressed in TRAIL-sensitive HTB-92 and HTB-93 cell lines, but not in TRAIL-resistant HTB-91 and HTB-94 cells. HTB-82 cells, which expressed the long (c-FLIP(L)) and short (c-FLIP(S)) splice variants of the FLICE-like inhibitory protein (FLIP), were resistant to TRAIL in spite of the presence of TRAIL-R1. TRAIL-R2,-R3,-R4 and osteoprotegerin (OPG) expression did not correlate with TRAIL sensitivity. Coincubation of TRAIL and doxorubicin led to the overexpression of TRAIL-R2 resulting in a synergistic effect of doxorubicin and TRAIL in TRAIL-sensitive cell lines and in the overcoming of TRAIL-resistance in all of the TRAIL-resistant cell lines, except HTB-91, which lacked caspase 8 expression. These data suggest that TRAIL, either as a single agent or in combination with cytotoxic agents, might represent a new treatment option for advanced STS, which constitutes a largely chemotherapy-resistant disease.