Induced Tumor Apoptosis Research Articles

A pH-Sensitive Prodrug Nanocarrier Based on Diosgenin for Doxorubicin Delivery to Efficiently Inhibit Tumor Metastasis.

BackgroundThe metastasis, one of the biggest barriers in cancer therapy, is the leading cause of tumor deterioration and recurrence. The anti.-metastasis has been considered as a feasible strategy for clinical cancer management. It is well known that diosgenin could inhibit tumor metastasis and doxorubicin (DOX) could induce tumor apoptosis. However, their efficient delivery remains challenging.PurposeTo address these issues, a novel pH-sensitive polymer-prodrug based on diosgenin nanoparticles (NPs) platform was developed to enhance the efficiency of DOX delivery (DOX/NPs) for synergistic therapy of cutaneous melanoma, the most lethal form of skin cancer with high malignancy, early metastasis and high mortality.Methods and ResultsThe inhibitory effect of DOX/NPs on tumor proliferation and migration was superior to that of NPs or free DOX. What is more, DOX/NPs could combine mitochondria-associated metastasis and apoptosis with unique internalization pathway of carrier to fight tumors. In addition, biodistribution experiments proved that DOX/NPs could efficiently accumulate in tumor sites through enhancing permeation and retention (EPR) effect compared with free DOX. Importantly, the data from in vivo experiment revealed that DOX/NPs without heart toxicity significantly inhibited tumor metastasis by exerting synergistic therapeutic effect, and reduced tumor volume and weight by inducing apoptosis.ConclusionThe nanocarrier DOX/NPs with satisfying pharmaceutical characteristics based on the establishment of two different functional agents is a promising strategy for synergistically enhancing effects of cancer therapy.

Shikonin induces tumor apoptosis in glioma cells via endoplasmic reticulum stress, and Bax/Bak mediated mitochondrial outer membrane permeability

Ethnopharmacological relevanceShikonin, one of the main active ingredients of Chinese herbal medicine Lithospermum erythrorhizon, has been widely used to treat various disease including virus infection and inflammation in clinical. Its anti-tumor activity has been recorded in “Chinese herbal medicine”. Recently, some studies about its anti-glioma effects have been reported. However, little is known about the molecular pharmacological activity of Shikonin in glioma. AimThis study aimed to systematically uncover and validate the pharmacological mechanism of Shikonin against glioma. Material and methodsNetwork pharmacology approach, survival analysis, and Pearson co-expression analysis were performed to uncover and test the pharmacological mechanisms of Shikonin in glioma. Apoptosis assay, Caspase-3 activity assay and immunoblot analysis were practiced to validate the mechanisms. ResultsNetwork pharmacology results suggested, anti-glioma effect of Shikonin by interfering endoplasmic reticulum (ER) stress-mediated tumor apoptosis targeting Caspase-3, and Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP) triggering cancer cell apoptosis. Survival analysis suggested the association of CASP3 with glioma (P < 0.05). Pearson correlation analysis indicated possible interaction of CASP3 with PERK through positive feedback regulation. Shikonin or in combination with 14G2a induced cell apoptosis in oligodendroglioma Hs683 cells in a dose-dependent manner with at a maximum apoptosis rate of 33%–37.5%, and 73%–77% respectively. Immunoblot analysis showed that Shikonin increased Caspase-3 activity to about 4.29 times, and increased 9 times when it combined with 14G2a. Shikonin increased also the expression levels of the proteins PERK and CHOP by about 4.4 and 5.6 folds, respectively, when it combined with 14G2a. ConclusionsThis study highlights the pharmacological mechanisms of Shikonin in the induction of tumor apoptosis in glioma cells.

Compound Opening Arrow Mixture exerts anti-tumor effects in a mouse model of breast cancer

Compound Opening Arrow Mixture (COAM) has demonstrated therapeutic effects in patients with breast cancer. We explored the underlying molecular mechanisms of COAM using a mouse model of breast cancer. Luciferase-labeled 4T1-Luc2 cells were inoculated into the breast pad of BALB/c-nu mice, which were divided into model group (saline), COAM (6 g/ml high-dose, 3 g/ml medium-dose, and 1.5 g/ml low-dose) groups, and low-molecular-weight heparin (LMWH, 1500 U/Kg) group. The number and distribution of 4T1-luc2 tumors were measured by an in vivo imaging system. Tumor cell apoptosis was measured through TUNEL and quantitating the expression of Caspase-3 mRNA and protein. Compared with the model group, in vivo tumor growth was lower in the LMWH- and COAM-treated groups. Tumor apoptosis was time-dependent and dose-dependent, as shown by a higher TUNEL apoptotic index and higher Caspase-3 mRNA and Caspase-3/cleaved-Caspase-3 proteins levels on the 14th day than the 7th day. The COAM high-dose group had the highest apoptotic index and the most activation of Caspase-3. Collectively, COAM significantly inhibits the growth of 4T1-luc2 breast cancer in mice and induces tumor apoptosis by activating Caspase-3, which provides a preliminary explanation of therapeutic effects of COAM.

Diplatin, a novel water-soluble platinum complex, inhibits lung cancer growth via augmentation of Fas-mediated apoptosis

Platinum drugs, such as cisplatin (DDP) and carboplatin (CBP), are the main drugs for the treatment of lung cancer, but their practical clinical application is limited by severe toxicity and acquired drug resistance. Our previous study has indicated that diplatin, [2-(4-(diethyl-amino)butyl)malonate-O,O’]-[(1R,2R)-cyclohexane-1,2-diamine N,N’] platinum (II) phosphate, a novel water-soluble platinum complex, could overcome DDP-resistant cells and was less toxic than comparable platinum drugs. In the present study, the effects and mechanisms of diplatin were further evaluated for its development as a novel anti-lung cancer platinum drug. Here, we found diplatin down-regulated the viability of H460 and LTEP-A-2 cells in a dose-dependent manner. Nude mice administrated with diplatin (30–120 mg/kg) via tail vein injection dose-dependently inhibited the growth of H460 and LTEP-A-2 xenograft tumors, whose action mainly correlated with the induction of tumor apoptosis. Particularly, the exposure of lung cancer cells or xenograft tumors to diplatin resulted in elevated Fas level, and knockdown of Fas ameliorated diplatin-induced cells apoptosis. Overall, we suggest that diplatin has potent anti-tumor activity, which probably acts through Fas-mediated signaling pathway.

Integration of Fe3O4 with Bi2S3 for Multi-Modality Tumor Theranostics.

The combination of reactive oxygen species (ROS)-induced chemodynamic therapy (CDT) and photothermal therapy (PTT) holds a promising application prospect for their superb anticancer efficiency. Herein, we created a novel Fe3O4@polydopamine (PDA)@bovine serum albumin (BSA)-Bi2S3 composite as a theranostic agent, by chemically linking the Fe3O4@PDA with BSA-Bi2S3 via the amidation between the carboxyl groups of BSA and the amino groups of PDA. In this formulation, the Fe3O4 NPs could not only work as a mimetic peroxidase to trigger Fenton reactions of the innate H2O2 in the tumor and generate highly cytotoxic hydroxyl radicals (•OH) to induce tumor apoptosis but also serve as the magnetic resonance imaging (MRI) contrast agent to afford the precise cancer diagnosis. Meanwhile, the PDA could prevent the oxidization of Fe3O4, thus supporting the long-term Fenton reactions and the tumor apoptosis in the tumor. The Bi2S3 component exhibits excellent photothermal transducing performance and computed tomography (CT) imaging capacity. In addition, the PDA and Bi2S3 endow the Fe3O4@PDA@BSA-Bi2S3 composite with an excellent photothermal transforming ability whichcould lead to tumor hyperthermia. All of these merits play the synergism with the tumor microenvironment and qualify the Fe3O4@PDA@BSA-Bi2S3 NPs for a competent agent in the MRI/CT-monitored enhanced PTT/CDT synergistic therapy. Findings in this research will evoke new interests in future cancer therapeutic strategies based on biocompatible nanomaterials.

Oncolytic adenovirus encoding LIGHT (TNFSF14) inhibits tumor growth via activating anti-tumor immune responses in 4T1 mouse mammary tumor model in immune competent syngeneic mice.

LIGHT, also known as tumor-necrosis factor (TNF) superfamily member 14 (TNFSF14), is predominantly expressed on activated immune cells and some tumor cells. LIGHT is a pivotal regulator both for recruiting and activating immune cells in the tumor lesions. In this study, we armed human telomerase reverse transcriptase (TERT) promoter controlled oncolytic adenovirus with LIGHT to generate rAd.Light. rAd.Light effectively transduced both human and mouse breast tumor cell lines in vitro, and expressed LIGHT protein on the surface of tumor cells. Both rAd.Null, and rAd.Light could replicate in human breast cancer cells, and produced cytotoxicity to human and mouse mammary tumor cells. rAd.Light induced apoptosis resulting in tumor cell death. Using a subcutaneous model of 4T1 cells in BALB/c mice, rAd.Light was delivered intratumorally to evaluate the anti-tumor responses. Both rAd.Light and rAd.Null significantly inhibited the tumor growth, but rAd.Light produced much stronger anti-tumor effects. Histopathological analysis showed the infiltration of T lymphocytes in the tumor tissues. rAd.Light also induced stronger cellular apoptosis than rAd.Null in the tumors. Interestingly, on day 15, compared to rAd.Null, there was a significant reduction of Tregs following rAd.Light treatment. rAd.Light significantly increased Th1 cytokine interleukin (IL)-2 expression, and reduced Th2 cytokines expression, such as transforming growth factor β (TGF-β) and IL-10 in the tumors. These results suggest rAd.Light induced activation of anti-tumor immune responses. In conclusion, rAd.Light produced anti-tumor effect in a subcutaneous model of breast cancer via inducing tumor apoptosis and evoking strong anti-tumor immune responses. Therefore, rAd.Light has great promise to be developed as an effective therapeutic approach for the treatment of breast cancer.

Chamaejasmin B Decreases Malignant Characteristics of Mouse Melanoma B16F0 and B16F10 Cells.

Chamaejasmin B (CHB), a natural biflavone isolated from Stellera chamaejasme L., has been reported to exhibit anti-cancer properties; however, its effect in melanoma cells is not clear. Here, we aimed to investigate the anticancer effect of CHB in mouse melanoma B16F0 and B16F10 cells. We found that CHB significantly suppressed cell proliferation and promoted cell cycle arrest at G0/G1 phase in B16F0 cells; it also induced cell differentiation and increased melanin content by increasing tyrosinase (TYR) activity and mRNA levels of melanogenesis-related genes in B16F0 cells. Meanwhile, wound closure, invasion, and migration of B16F0 and B16F10 cells were dramatically inhibited. Moreover, CHB significantly increased ROS levels and decreased ΔΨm, resulting in B16F0 and B16F10 cell apoptosis. Finally, in vivo studies showed that CHB inhibited tumor growth and induced tumor apoptosis in a mouse xenograft model of murine melanoma B16F0 and B16F10 cells. Overall, CHB decreases malignant characteristics and may be a promising therapeutic agent for malignant melanoma cells via multiple signaling pathways.

A multifunctional MOF-based nanohybrid as injectable implant platform for drug synergistic oral cancer therapy

Toward eradicating tumor, it is crucial to facilitate the sustained delivery of encapsulated drugs and render enhancements of therapeutic effectiveness. Local cancer therapy with combined drugs has emerged as a promising therapeutic strategy. Herein, we put forward to construct a hybrid nanocomposite in which metal-organic frameworks (MOFs) were integrated with thermosensitive hydrogels to devise an injectable implant. Doxorubicin (Dox) and celecoxib (Cel) were coloaded into the system for localized oral cancer therapy (Dox/Cel/MOFs@Gel). In view of the antiangiogenetic activity of Cel, Cel was in alliance with Dox in order to improve treatment efficacy. The particle size, morphological characterization from SEM and TEM, stability, drug loading efficiency, drug release behaviors in vitro of Dox/Cel/MOFs@Gel were examined. The efficacy of Dox/Cel/MOFs@Gel in oral cancer cell lines as well as cell internalization was evaluated. This medical platform exhibited a high capacity for drug loading, steady and pH-responsive release of dual drugs, and enhanced toxic effects against oral cancer cells (KB and SCC-9) in vitro. The nanocomposites displayed outstanding tumor inhibition efficacy in vivo, inducing tumor apoptosis and regulating tumor angiogenesis due to the synergistic effects of Dox and Cel. It was found that this local treatment resulted in considerably lower systemic toxicity and no apparent injury to the other organs. The biocompatibility test of the MOFs indicated reasonable biosafety as no evidence of persistent toxicity in vivo was observed. The injectable nanocomposite (Dox/Cel/MOFs@Gel) possesses unique biological abilities in terms of pH-responsiveness, antitumor efficacy, biocompatibility, and simultaneously release hydrophobic and hydrophilic drug at the oral tumor site, suggesting that this nanocomposite is a promising vehicle for local oral cancer treatment.

Quercetin promotes the survival of granulocytic myeloid-derived suppressor cells via the ESR2/STAT3 signaling pathway

Quercetin is a natural product that has been shown to induce tumor apoptosis and necrosis through multiple mechanisms. Tumor-induced myeloid-derived suppressor cell (MDSC) expansion negatively regulates the immune response by inhibiting T cell function through signal transducer and activator of transcription 3 (STAT3) activation, thereby facilitating tumor escape from host immune surveillance. Thus MDSC is an attractive target for cancer immunotherapy to enhance cytotoxic T cell responses. However, the effects of quercetin on MDSC are poorly understood. Here, we demonstrate that quercetin treatment enhanced mouse- and human- derived granulocytic-myeloid-derived suppressor cells (G-MDSC) survival and promoted the secretion of T cell-suppressive factors in vitro. Bioinformatics analysis further showed that quercetin was highly correlated with the estrogen receptor signaling pathway, which was confirmed by quantitative reverse transcription-polymerase chain reaction and flow cytometric analysis. These findings highlight the potential advantages and feasibility of quercetin in reinforcing the suppressive property of G-MDSC. Thus impact of G-MDSC should be taken into consideration when quercetin is applied to tumor therapy.

The Interleukin-33/ST2 axis promotes glioma mesenchymal transition, stemness and TMZ resistance via JNK activation.

IL-33 is an important member of the IL-1 family which has pleiotropic activities in innate and adaptive immune responses. Recently, some researchers have focused on the function of cellular immunity in the development of tumor. The biological role of IL-33 in glioma is poorly understood. In this study, we showed that glioma cells and tissues expressed higher levels of IL-33 and its receptor ST2 compared to normal brain. Clinically, IL-33 expression was associated with poor survival in patients with glioma. Administration of human IL-33 enhanced cell migration, invasion, epithelial to mesenchymal transition and stemness. Anti-ST2 blocked these effects of IL-33 on tumor. Mechanistically, IL-33 activated JNK signaling pathway via ST2 and increased the expression of key transcription factors that controlled the process of EMT and stemness. Moreover, IL-33 prevented temozolomide induced tumor apoptosis. Anti-ST2 or knockdown IL-33 increased the sensitivity of tumor to temozolomide. Thus, targeting the IL-33/ST2 axis may offer an opportunity to the treatment of glioma patients.

Metal-Organic Frameworks@Thermosensitive Hydrogel as Injectable Implant for Dual Drug Synergistic Oral Cancer Therapy

Local cancer therapy with multiple drugs has emerged as a novel therapeutic approach for facilitating the sustained delivery of therapeutic molecules that effectively suppress tumor growth. In this study, we developed a hybrid nanocomposite in which metal-organic frameworks (MOFs) were integrated with thermosensitive hydrogels to devise an injectable implant. Doxorubicin (Dox) and celecoxib (Cel) were coloaded into the system for localized oral cancer therapy (Dox/Cel/MOFs@Gel). This medical platform exhibited a high capacity for drug loading, steady and pH-responsive release of dual drugs, and enhanced toxic effects against oral cancer cells (KB and SCC-9) in vitro. The nanocomposites displayed outstanding tumor inhibition efficacy in vivo, inducing tumor apoptosis and regulating tumor angiogenesis due to the synergistic effects of Dox and Cel. It was found that this local treatment resulted in considerably lower systemic toxicity and no apparent injury to the other organs. The biocompatibility test of the MOFs that constructed the nanocomposite indicated reasonable biosafety in vivo as no evidence of persistent toxicity was observed. The results of this study demonstrate that the injectable nanocomposite (Dox/Cel/MOFs@Gel) possesses unique biological abilities in terms of pH-responsiveness, antitumor efficacy, and biocompatibility, suggesting that the nanocomposite is a promising vehicle for local oral cancer treatment.

TRIM59: A membrane protein expressed on Bacillus Calmette-Guérin-activated macrophages that induces apoptosis of fibrosarcoma cells by direct contact

Bacillus Calmette-Guérin (BCG)-activated macrophages (BAMs) have anti-tumor effects, especially on fibrosarcoma cells. However, the mechanism governing this process has not been elucidated to date. TRIM59 is an up-regulated membrane protein expressed on the surface of BAMs. In this study, we found that up-regulated TRIM59 macrophages exhibited excellent growth inhibition on MCA207 fibrosarcoma and induced tumor apoptosis. Moreover, TRIM59 enhanced macrophage infiltration and increased the M1 phenotype macrophages inside the tumor. Furthermore, the cytotoxic T cells and B cells in the spleen and lymphnode have not been affected by TRIM59. These results showed that macrophages expressing TRIM59 exhibited the main cytotoxic effect on tumors. In vitro, we co-cultured TRIM59 up-regulated macrophages fixed with 1% paraformaldehyde or cell culture supernatant and tumor cells. We found that the killing activities of macrophages decreased after treatment with anti-TRIM59 antibody, and the supernatant of TRIM59 up-regulated macrophages had no tumoricidal effect on fibrosarcoma cells, which demonstrated that TRIM59 may be involved in tumoricidal effects via cell-cell contact. In addition, the PI3K-Akt pathway of MCA207 co-cultured with macrophages highly expressing TRIM59 was significantly inhibited, whereas the activation of the PI3K-Akt pathway in MCA207 was not affected after co-culture with TRIM59-CKO macrophages. These results define a vital role of TRIM59 as an anti-tumor effector molecule of BAMs and suggest a new therapeutic target for the treatment of fibrosarcoma.

Soluble fms-like tyrosine kinase-1-enriched exosomes suppress the growth of small cell lung cancer by inhibiting endothelial cell migration.

BackgroundPrevious studies have reported that soluble fms‐like tyrosine kinase‐1 (sFlt‐1) possesses anti‐tumor effects by inhibiting angiogenesis in many cancers. Exosomes can be engineered as delivery vehicles for transferring functional biomolecules, such as proteins, lipids, and nucleic acids (DNA, mRNA, and miRNA) to target cells to affect inflammation, apoptosis, and angiogenesis. The purpose of this study was to investigate whether exosomes can function as efficient carriers of sFlt‐1 in vitro and in vivo, to play a role in SCLC therapy.MethodsWe adopted three different methods: TEM, NTA and western blot analysis to characterize the cell‐derived exosomes from NCI‐H69 SCLC cell line and normal bronchial epithelial BEAS‐2B cell line. we next explored the effects of these exosomes on HUVE cell proliferation and migration in vitro.To verify sFlt‐1‐loaded exosomes suppress the tumor growth in vivo,we established subcutaneous xenografts in nude mice using the NCI‐H69 cell line.ResultsWe observed that NCI‐H69‐exo significantly increased human umbilical vein endothelial cells (HUVEC) migration compared to BEAS‐2B‐exo in vitro and in vivo. sFlt‐1 protein expression was statistically higher in BEAS‐2B‐exo than NCI‐H69‐exo. sFlt‐1 protein or sFlt‐1‐enriched exosomes can inhibit the migration of HUVECs. Furthermore, sFlt‐1‐enriched exosomes exhibited higher inhibition efficacy on pro‐angiogenesis of NCI‐H69‐exo in comparison with the same concentration of sFlt‐1 protein. Intriguingly, sFlt‐1‐loaded exosomes showed marked anti‐tumor activity by inhibiting the growth of NCI‐H69 tumor xenografts. CD31 staining revealed that sFlt‐1‐loaded exosomes significantly reduced the vascular density of experimental mice. sFlt‐1‐loaded exosomes markedly induced tumor apoptosis and inhibited tumor cell proliferation in mice.ConclusionExosomes from a SCLC cell line contain low levels of sFlt‐1 and significantly increased the migration of HUVECs. SFlt‐1‐enriched exosomes can inhibit NCI‐H69‐exo‐induced HUVEC migration. Exosomes enriched in sFlt‐1 have the potential to be effective therapeutic agents for SCLC.

Enhancement mitochondrial apoptosis in breast cancer cells by paclitaxel-triphenylphosphonium conjugate in DNA aptamer modified nanoparticles

Mitochondria is a major regulator of inducing tumor apoptosis and it is as a prime target for chemotherapy. Anticancers need to access specific tumor cells and also pass through different barriers such as outer and inner membranes of mitochondria. Targeted accumulation of paclitaxel (PTX) within the mitochondria was achieved by direct conjugation of PTX with triphenylphosphonium (TPP) as a mitochondrial membrane-permeable cation. PTX-TPP prodrug was synthesized by pH-sensitive ester bond between the hydroxyl function of PTX and the carboxylic function of TPP. This ester bond is only cleaved by the mitochondrial aldehyde dehydrogenase. Prodrug was loaded in the albumin nanoparticles by nab-technology. The surface aptamer modification of nanoparticles was done by MUC1 DNA aptamer in order to targeting of tumor cells overexpressing MUC1. The yield of PTX-TPP conjugation was 81% and the particle size of final nanoparticles was 139 nm. The aptamer-modified nanoparticles significantly increased the cellular uptake to 99.9%, and demonstrated good mitochondrial uptake in the confocal fluorescence images leading to apoptosis of MCF-7 breast cancer cells. Early apoptosis and late apoptotic/necrotic phase of cells after 24 h were 26.4% and 25.3%, respectively, compared to 5.27% and 4.74% for free PTX. The targeted nanoparticles exhibited the superior in vitro anticancer efficacy of up to 17.8 fold higher than free PTX after 72 h. This multi-targeting strategy induced the mitochondrial apoptosis to killing tumor cells.

Stress-Induced, p53-Mediated Tumor Growth Inhibition of Melanoma by Modulated Electrohyperthermia in Mouse Models without Major Immunogenic Effects.

Modulated electrohyperthermia (mEHT), an innovative complementary technique of radio-, chemo-, and targeted oncotherapy modalities, can induce tumor apoptosis and contribute to a secondary immune-mediated cancer death. Here, we tested the efficiency of high-fever range (~42 °C) mEHT on B16F10 melanoma both in cell culture and allograft models. In vivo, mEHT treatment resulted in significant tumor size reduction when repeated three times, and induced major stress response as indicated by upregulated cytoplasmic and cell membrane hsp70 levels. Despite the increased PUMA and apoptosis-inducing factor 1, and moderate rise in activated-caspase-3, apoptosis was not significant. However, phospho-H2AX indicated DNA double-strand breaks, which upregulated p53 protein and its downstream cyclin-dependent kinase inhibitors p21waf1 and p27kip. Combined in vitro treatment with mEHT and the p53 activator nutlin-3a additively reduced cell viability compared to monotherapies. Though mEHT promoted the release of damage-associated molecular pattern (DAMP) damage signaling molecules hsp70, HMGB1 and ATP to potentiate the tumor immunogenicity of melanoma allografts, it reduced MHC-I and melan-A levels in tumor cells. This might explain why the number of cytotoxic T cells was moderately reduced, while the amount of natural killer (NK) cells was mainly unchanged and only macrophages increased significantly. Our results suggest that mEHT-treatment-related tumor growth control was primarily mediated by cell-stress-induced p53, which upregulated cyclin-dependent kinase inhibitors. The downregulated tumor antigen-presenting machinery may explain the reduced cytotoxic T-cell response despite increased DAMP signaling. Decreased tumor antigen and MHC-I levels suggest that natural killer (NK) cells and macrophages were the major contributors to tumor eradication.

DNA-Methylation-Caused Downregulation of miR-30 Contributes to the High Expression of XPO1 and the Aggressive Growth of Tumors in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma is one of the most aggressive cancers, with high mortality in the United States. One of the important signal transduction proteins involved in the regulation of pancreatic cancer’s aggressive progression is the nuclear export protein (XPO1). High expression of XPO1 has been found in pancreatic, lung, breast and other cancers and lymphomas with a poor prognosis of patients with tumors and high proliferative activity of cancer cells. Because XPO1 exports multiple tumor suppressor proteins simultaneously from the nucleus, the inhibition of XPO1 may retain multiple tumor suppressors in the nucleus, resulting in the suppression of cell proliferation and the induction of apoptosis in tumors. In this study, we found that the high expression of XPO1 in pancreatic cancer cells could be, in part, due to the methylation of the miR-30 gene, leading to the low expression level of the miR-30 family. By co-transfection of the XPO1 3′-UTR-Luc target vector with miR-30 mimic, we found that XPO1 is a direct target of the miR-30 family. We also observed that the enforced expression of the miR-30 family inhibited the expression of XPO1, resulting in the suppression of pancreatic cancer growth both in vitro and in vivo. These findings could help to design a novel therapeutic strategy for the treatment of pancreatic cancer by introducing miR-30 into cancer cells.

PH and Thermal Dual-Sensitive Nanoparticle-Mediated Synergistic Antitumor Effect of Immunotherapy and Microwave Thermotherapy.

Cationic anticancer peptides, which can induce tumor cell immunogenic death and further promote systemic tumor-specific immune responses, have offered a promising solution to relieve the tumor immunosuppressive microenvironment. However, peptide drugs are easily degraded and lack of targeting ability when administered systemically, leading to limitations in their applications. Herein, we report a pH and thermal dual-sensitive bovine lactoferricin-loaded (one of the most widely studied cationic anticancer peptides) nanoparticles, which simultaneously exhibited antitumor and immune cell activated effects when applied with microwave thermotherapy, an auxiliary method of immunotherapy. The bovine lactoferricin could be delivered to the tumor site by nanoparticles, be immediately released from nanoparticles in the acidic environment of lysosomes and the thermal condition caused by microwave radiation, and ultimately induce tumor apoptosis with the release of damage-associated molecular patterns (DAMPs). It is worth noting that the strategy of bovine lactoferricin-loaded nanoparticles intravenous injection combined with local microwave thermotherapy not only showed excellent efficacy in relieving tumor growth but also resulted in strong antitumor immunities, which was due to the released bovine lactoferricin under stimulating conditions, and the pool of tumor-associated antigens generated by tumor destruction. In conclusion, this work presents a strategy for tumor treatment based on dual-sensitive bovine lactoferricin-loaded nanoparticles combined with microwave thermotherapy, which may provide a solution for cationic anticancer peptides delivery and improving antitumor immune responses.

Abstract 2206: Anti-tumor activity of infigratinib, a potent and selective inhibitor of FGFR1, FGFR2 and FGFR3, in FGFR fusion-positive cholangiocarcinoma and other solid tumors

Abstract Abnormal expression and constitutive activation of receptor tyrosine kinases, such as ALK, ROS1 and TRK, as a result of gene rearrangements have been clinically validated as therapeutic targets for cancer. Recently, fusions involving the FGFR family, especially FGFR1, FGFR2 and FGFR3, have been identified in diverse solid tumors such as cholangiocarcinoma, glioblastoma, bladder, lung, breast, thyroid and prostate cancers. FGFR fusions are oncogenic drivers that ligand-independently activate the receptor kinases and their downstream signaling pathways, leading to uncontrolled cell proliferation and invasion. Infigratinib (BGJ398) is an oral and selective inhibitor of FGFR. At biochemical and cellular levels, infigratinib selectively inhibits the activity of FGFR1, FGFR2 and FGFR3 with low nM potency, while sparing FGFR4, VEGFR2 and other kinases. In FGFR fusion-positive xenograft models, including cell line-derived and patient-derived models representing diverse tumor histologies, infigratinib treatment leads to significant tumor growth inhibition and regression at clinically achievable exposure levels. The anti-tumor effect of infigratinib is accompanied by inhibition of downstream signaling pathways and induction of tumor apoptosis. Clinically, in an open-label phase II trial, infigratinib demonstrated a cORR of 39.3% in FGFR2 fusion-positive cholangiocarcinoma patients who had received one or fewer prior chemotherapy regimens. Additionally, RECIST responses were achieved in non-cholangiocarcinoma solid tumors tested positive for FGFR fusions. In conclusion, the available preclinical and clinical data demonstrate the potential of infigratinib as an effective treatment for patients with FGFR fusion-positive tumors, regardless of the fusion partners or the origin of tissue. On the basis of these data there is a clear rationale for performing tumor-agnostic clinical trials in molecularly defined cancers to maximally benefit patients with serious and life-threatening diseases. Citation Format: Gary Li, Melanie Krook, Sameek Roychowdhury, Francesca Avogadri, Yining Ye, Susan Moran. Anti-tumor activity of infigratinib, a potent and selective inhibitor of FGFR1, FGFR2 and FGFR3, in FGFR fusion-positive cholangiocarcinoma and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2206.

Abstract 1535: CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1 exhibits potent anti-tumor activity in DLK-1 expressing tumor xenograft models

Abstract Delta-like 1 homolog (DLK-1) is a type I transmembrane protein with 6 tandem EGF-like motifs in extracellular region. In fetal development, DLK-1 is expressed at high levels in undifferentiated, highly proliferative tissues, such as, liver, kidney, skeletal muscle and brain. In the fetal liver, it has reported that DLK-1 positive hepatoblasts have a differentiation capability toward hepatocyte and cholangiocyte in vitro. In adult tissues, its expression is restricted in tissues such as adrenal grand and hypophysis. On the other hand, overexpression of DLK-1 in cancers have been reported in several cancer types, such as hepatocellular carcinoma (HCC), neuroblastoma, rhabdomyosarcoma, small cell lung carcinoma, myelodysplastic syndrome. Interestingly, the expression of DLK-1 is overlapped with other hepatic progenitor cell markers such as EpCAM in HCC and DLK-1 has been suggested to be a marker of cancer stem cells in HCC. According to these selective expressions of DLK-1 in cancer tissues as well as cancer stem cells in HCC, DLK-1 is supposed to be a potential target for antibody-based therapy. CBA-1205 is a novel humanized antibody (IgG1/κ) targeting DLK-1 which was glycol-engineered by GlymaxX® to potentiate antibody-dependent cellular cytotoxicity (ADCC) activity. The purpose of this study is to evaluate the anti-tumor activity of CBA-1205 in multiple human cancer cell lines in vitro and xenograft models in vivo. In vitro, CBA-1205 showed potent ADCC activity in several DLK-1 positive human cancer cell lines for different cancer types. In vivo, CBA-1205 exhibited potent anti-tumor activity in several human cancer xenograft mouse models. In a Hep3B HCC xenograft model, we evaluated dose-dependent efficacy of CBA-1205 at a dose of 0.1, 0.3, 1, 3 and 10 mg/kg by the intraperitoneally injection. The tumor growth was inhibited in a dose-dependent manner and the inhibition rate at the dose of ≥ 1 mg/kg was more than 90% compared to isotype human IgG1 control. At 10 mg/kg, CBA-1205 treatment resulted in tumor regression in all mice and 4 complete tumor elimination out of 8 mice was observed. Single injection of 10 mg/kg CBA-1205 induced tumor apoptosis and reduced the CD31 positive tumor micro vessel at 24 and 48 h after the treatment. Similar anti-tumor activity of CBA-1205 was exhibited in other xenograft model of HCC-derived HepG2 and neuroblastoma-derived SK-N-FI, BE2-C and Kelly. In conclusion, we demonstrated that a novel glycoengineered humanized anti-DLK-1 antibody, CBA-1205, exerts potent anti-tumor activity in vitro and in vivo in multiple models and could be a novel treatment option for DLK-1 expressing cancer such as HCC. First-in-human Phase I study of CBA-1205 is planned to be initiated in Q1 2020. Citation Format: Koji Nakamura, Kota Takahashi, Izumi Sakaguchi, Zhang Lingyi, Hiroyuki Yanai, Toru Kanke. CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1 exhibits potent anti-tumor activity in DLK-1 expressing tumor xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1535.

Generation of CART cells targeting oncogenic TROP2 for the elimination of epithelial malignancies

Background & Aim TROP2 (also referred to as Tacstd2) is a membrane-associated glycoprotein highly expressed in many epithelial cancers, including oral, head-and-neck, thyroid, lung, esophageal, gastric, colorectal, pancreatic, breast, renal, uterine, cervical, ovarian, prostate, and glioma, and expression of TROP2 negatively correlates with prognosis and survival in many of these malignancies. TROP2 signaling supports tumor growth and progression through the induction of pathways controlling cell cycle progression, invasion and metastasis, proliferation, angiogenesis, cell growth and survival. In addition, tumor release of TROP2-containing exosomes can promote the migration of TROP2-negative tumor cells. Here, we generated several TROP-2-targeting CAR molecules and evaluated their activity as CART cells against various epithelial tumor cell lines. Methods, Results & Conclusion Single-chain variable fragments targeting TROP2 were produced through domain sequencing of hybridomas and through other publically available sources and cloned into lentiviral transfer plasmids containing second-generation CAR backbones. Transduction of human T cells routinely leads to the expression of >50% CAR+ cells, including the generation of TROP2-CART cells from T cells of a mTNBC patient. We evaluated interferon-gamma production as well as cytotoxicity of TROP2-CART cells cultured with TROP2+ breast, prostate and pancreatic tumor cells to choose a lead TROP2-CAR. In addition, we generated an isogenic TROP2- prostate cancer through Cas9-based gene editing and observed loss of cytotoxicity, demonstrating specificity of the TROP2-CART. However, TROP2-CART can kill TROP2- cells in the presence of TROP2+ cells, a feature also observed by CART19 when cultured with CD19+ and CD19- cells, consistent prior reports that activated CART cells induce tumor apoptosis through death receptors. Prior reports demonstrated that blockade of death receptor ligands abolished the non-specific cytotoxicity of CART19 cells, but this approach provided only partial blockade of TROP2-CART cytotoxicity. Instead, we describe and demonstrate a new mechanism by which transfer of TROP2+ tumor exosomes to TROP2- tumor cells increases the proportion of tumor targetable by TROP2-CART and may allow TROP2-CART to overcome the challenge of antigen heterogeneity in solid tumors.