Growth Inhibition Rate Research Articles

Abstract 1631: Combination of olverembatinib (HQP1351) with BCL-2 inhibitor lisaftoclax (APG-2575) overcomes resistance in gastrointestinal stromal tumors (GISTs)

Abstract GISTs are common malignant mesenchymal tumors that occur in the GI tract, with an estimated incidence rate of 1% to 2%. About 75% to 80% of patients with GIST have mutations in the KIT gene, while 5% to 10% have mutations in the platelet-derived growth factor receptor α (PDGFRA) gene. Tyrosine kinase inhibitors (TKIs) offer patients an improved quality of life but increased pharmacological resistance to TKIs is often observed. BCL-2 is expressed in >80% of GISTs, and amplification of BCL-2 and BCL-xL are common features associated with disease progression. One approach to enhance GIST eradication is to concurrently inhibit oncogenic KIT signaling while actively engaging apoptotic pathways. Olverembatinib (HQP1351) is a new, third-generation TKI that targets BCR-ABL1, KIT and PDGFRA and is currently in development for relapsed or refractory chronic myeloid leukemia and GIST. Lisaftoclax (APG-2575) is a selective BCL-2 inhibitor under development for hematologic malignancies. The purpose of this study was to evaluate whether combining a BCL-2 inhibitor, lisaftoclax, with olverembatinib enhances treatment effect on imatinib-resistant GISTs. Compared to imatinib-sensitive GIST cell lines (GIST882 and GIST-T1), increased BCL-2 protein expression was observed in imatinib-resistant GIST cells, particularly in GIST430, GIST48, and GIST48B lines. Cell-based antiproliferation studies showed additive activity of olverembatinib and lisaftoclax in imatinib-resistant GIST cells. In vivo studies using imatinib-resistant GIST430 cell-derived xenograft models further revealed that coadministration of olverembatinib at 15 mg/kg (every other day for 3 weeks) with lisaftoclax at 50 mg/kg (daily for 3 weeks) resulted in enhanced antitumor effects compared to either agent alone. Tumor growth inhibition (TGI) rates for combined treatment with olverembatinib and lisaftoclax reached 76.8%, which was superior to the TGI rate of either agent alone: 57.6% and 31.2% for olverembatinib and lisaftoclax, respectively. Regarding putative mechanisms, olverembatinib treatment was shown to downregulate BCL-xL and MCL-1 protein expression, possibly via inhibition of KIT signal pathway and downstream signal transducer and activator of transcription 3 (STAT3). Lisaftoclax triggered apoptosis by disrupting the BCL-2:BIM complex. Therefore, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly augmented cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1), thus triggering apoptosis and subsequently enhancing antitumor effects. Our results demonstrate that olverembatinib and BCL-2 inhibitor lisaftoclax have additive antitumor effects in imatinib-resistant GIST. This novel dual approach may have the potential for treating patients with GISTs whose disease has progressed after treatment with TKIs. Citation Format: Ping Min, Guangfeng Wang, Eric Liang, Bingxing Wu, Huidan Yu, Dajun Yang, Yifan Zhai. Combination of olverembatinib (HQP1351) with BCL-2 inhibitor lisaftoclax (APG-2575) overcomes resistance in gastrointestinal stromal tumors (GISTs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1631.

Abstract 1632: MDM2 inhibitor alrizomadlin (APG-115) promotes antitumor activity of mitogen-activated protein kinase (MAPK) inhibitors in uveal melanoma

Abstract Uveal melanoma (UM) is the commonest primary intraocular malignancy, yet its molecular pathogenesis is poorly understood. Most UM cases have activating mutations in genes encoding G protein subunits alpha Q or 11, leading to activation of downstream effectors. These include protein kinase C, mitogen-activated protein kinases (MAPK1/3; also termed extracellular signal-regulated kinase 2/1 [ERK2⁄ERK1], respectively), and yes-associated protein, suggesting a rationale for therapeutically targeting these related pathways. Genetic analyses show that TP53 is infrequently mutated in UM, but associated pathways may be functionally inactivated. Ubiquitination-mediated degradation of p53 activates MAPK signaling, such that, active p53 may promote suppression of MAPK signaling. Alrizomadlin (APG-115) is a small molecule targeting p53/MDM2 that is in clinical development for solid and hematologic cancers. This study evaluated the antitumor effect of alrizomadlin, alone or combined with other targeted therapies, in preclinical models of UM. In cell-based antiproliferation assays of many UM cell lines, alrizomadlin exerted moderate growth inhibitory effects (IC50 = 0.151-1.857μM). When combined with inhibitors against mitogen-activated protein kinase kinase (MEK) [trametinib and selumetinib], mammalian target of rapamycin (mTOR) [rapamycin], focal adhesion kinase (FAK) [defactinib and Ascentage’s APG-2449, which targets FAK/ALK/ROS1], and MEK/rapidly accelerated fibrosarcoma [RAF] [VS-6766], alrizomadlin demonstrated enhanced antiproliferative activity. In a UM xenograft model (MP41), alrizomadlin showed dose-dependent antitumor activity when administered at 25, 50, and 100 mg/kg, with tumor growth inhibition (TGI) rates of 24.8%, 38%, and 39.1%, respectively. In this model, synergistic antitumor effects were observed when alrizomadlin was combined with selumetinib or APG-2449, with TGI rates of 65.4% and 47.5%, respectively. Mechanistically, alrizomadlin likely increased p53, MDM2, and downstream p21 in MP41 cells. Importantly, alrizomadlin inhibited ERK phosphorylation and enhanced selumetinib inhibition in a dose- and time-dependent manner. Prior studies indicated that FAK can suppress p53 by enhancing MDM2-dependent p53 ubiquitination. In this study, APG-2449 showed dose-dependent inhibition of FAK phosphorylation and upregulation of p53. Alrizomadlin also inhibited phosphorylation of FAK and downstream ERK. Therefore, the combination of alrizomadlin and APG-2449 was synergistic. Combining MEK and FAK inhibitors also markedly augmented caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage (apoptosis hallmarks) and enhanced observed antitumor effects. In conclusion, our results demonstrate the potential utility of combining alrizomadlin with MAPK pathway inhibitors to treat patients with UM. Citation Format: Guangfeng Wang, Eric Liang, Ping Min, Dajun Yang, Yifan Zhai. MDM2 inhibitor alrizomadlin (APG-115) promotes antitumor activity of mitogen-activated protein kinase (MAPK) inhibitors in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1632.

Abstract 6433: A novel bacterial strain, CJRB-101, induces anti-cancer effects by repolarization of M2 to CXCL9 and CXCL10 dual expressing M1 macrophages in humanized non-small cell lung cancer mice models

Abstract Backgrounds: Live biotherapeutic products (LBPs) emerged as potential therapeutics to overcome the limitation of ICIs. This research shows that CJRB-101, a novel bacterial strain, can improve anti-tumor effects in synergy with pembrolizumab in non-small cell lung cancer (NSCLC). Objectives and Methods: Tumors from NSCLC patients (anti-PD-1 refractory and resistant) were transplanted into Hu-CD34-NSG to establish humanized patient-derived xenograft (PDX) mice models. Five models (YHIM-2003, 2004, 2009, 2010 and 2014) were treated with CJRB-101 at low (5 × 107 CFU) or high (109 CFU) doses, or with pembrolizumab (10 mg/kg, i.p., Q5D) or in combination. Tumor growth inhibition (TGI) rate was measured. Tumor microenvironment (TME) was analyzed using multiplex IHC, flow cytometry and single cell RNA sequencing. Ex-vivo assays were performed to validate in silico findings. Results: Tumor in PDX models was unresponsive to pembrolizumab alone, however, in combination with CJRB-101 effectively suppressed tumor growth. The synergy was highlighted in YHIM-2009 where TGI was 10-fold higher (56%) than pembrolizumab group (5%). Immune profiling revealed that macrophages may be responsible for the anti-tumor effects of CJRB-101. IHC showed significantly increased antigen presenting specialized DCs (CD16+CD68−CD11c+) and granzyme B+ CD8+ T cells in the tumor by CJRB-101 compared to pembrolizumab (p<0.01). This suggested that CJRB-101 induced infiltration of cytotoxic CD8 T cells into the tumor nest by enhancing antigen presenting machinery. Trajectory analysis showed that CJRB-101 induced repolarization of M2 to M1 macrophages, characterized by high expression of CXCL9/10. CXCL9+/10+ M1 macrophages were comparatively more abundant in the combination group (23.11%) than the pembrolizumab group (0.91%). CXCL9/CXCL10 expression in macrophages was higher in the CJRB-101 group compared to the pembrolizumab group (p<0.0001). The combination group (10.84%) had a higher relative abundance of CD8+ T cells compared to the pembrolizumab group (1.58%) and higher IFNγ expression in CD8+ T cells compared to the pembrolizumab group (p=0.0152), suggesting that CJRB-101 repolarized macrophages and recruited active CD8+ T cells. Co-culture assays using bone marrow-derived macrophages validated that CJRB-101 drove differentiation towards F4/80+ or MHC II+ expressing M1 macrophage (p<0.0001) and repolarized existing M2 (CD206+) to M1 (p=0.0002). Conclusion: Combination treatment of CJRB-101 with anti-PD-1 showed synergistic anti-tumor effects via repolarization of M2 to M1 macrophages, leading to activation of CD8+ T cells in TME. Especially, CXCL9+/10+ M1 macrophage playing a key role in TGI induced by CJRB-101 in NSCLC models. Findings from this study provided rationale for clinical investigation of CJRB-101. Citation Format: Arim Min, Chun-bong Synn, Seong-san Kang, Bo-eun Kwon, Junwon Yang, Hyunkyung Park, Jieun Im, Hyunjeong Kim, Sujeong Beak, Dong Kwon Kim, Jii Bum Lee, Hyeonseok Oh, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Sungwoo Lee, Mi Ran Yun, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Ji Yun Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. A novel bacterial strain, CJRB-101, induces anti-cancer effects by repolarization of M2 to CXCL9 and CXCL10 dual expressing M1 macrophages in humanized non-small cell lung cancer mice models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6433.

Abstract 1818: Developing an autologous humanized mouse model to assess responses to novel immunotherapeutics

Abstract Introduction: The development and FDA approval of drugs targeting the PD-1/PD-L1 axis has greatly shifted the paradigm of immunotherapy and has improved the clinical outcome for patients diagnosed with advanced melanoma. Despite the recent advancement of immune checkpoint inhibitors, more than 50% of patients still fail to show a sustained response to therapy. Therefore, there is a critical need to develop therapies specific to novel immune targets as well as combination drug regimens. Current immunocompetent preclinical models do not adequately model the responses to immunotherapy, reflecting the urgent need to develop models with greater translational utility. To address this need, we developed a humanized xenograft melanoma model engrafted with autologous peripheral blood lymphocytes (PBLs) to evaluate the utility of nemvaleukin alfa, an engineered IL-2 cytokine which preferentially activates and expands cytotoxic NK cells and antigen-experienced CD8 T cells. Methods: Melanoma-bearing mice were injected intravenously with matched melanoma patient PBLs and treated for four cycles (1 cycle/week) with anti-PD-1, nemvaleukin, or rhIL-2. Tumor growth was monitored, and correlative analyses were done on blood, spleen, and tumors by flow cytometry after 4 cycles of treatment. Results: This humanized melanoma mouse model exhibited good T cell engraftment and was characterized by expression of the proliferative marker Ki-67 in 50-60% of CD8+ and CD4+ T cell populations. Treatment with nemvaleukin resulted in significant tumor growth inhibition rates (TGIs) of an average of 48% compared to vehicle-treated mice. In addition, nemvaleukin promoted expansion of CD8+ and CD4+ T cells, without expansion of CD4+ regulatory T cells, which are known to suppress immunological responses. Compared to rhIL-2 and vehicle, nemvaleukin treatment delayed tumor growth and enhanced survival with a median survival time of 56 days compared to 21 and 50 days, respectively. Nemvaleukin-treated mice had higher CD45+ cell engraftment in spleens and tumors compared to anti-PD-1 treatment, indicating that nemvaleukin boosts T cell proliferation and expansion. Of note, we observed better antitumor efficacy in response to nemvaleukin or anti-PD-1 when mice were engrafted with tumors from patients who responded to checkpoint immunotherapy with TGIs of 36% and 51%, respectively, compared to those who did not with TGIs of 16% and 12%, respectively. These data indicate that treatment responses in our humanized model correlates with clinical outcomes. Conclusion: In this all autologous humanized melanoma mouse model, nemvaleukin treatment not only enhances T cell engraftment, it significantly augments anti-tumor efficacy. These data support the use of this personalized melanoma xenograft model to analyze the underlying immune responses in responders and non-responders to novel immunotherapies. Citation Format: Yee Peng Phoon, Jared E. Lopes, Lukas W. Pfannenstiel, C. Marcela Diaz-Montero, Ye F. Tian, Marc S. Ernstoff, Pauline Funchain, XiaoXia Li, Heather C. Losey, Raymond J. Winquist, Brian R. Gastman. Developing an autologous humanized mouse model to assess responses to novel immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1818.

Abstract 3242: Efficacy evaluation by novel humanized mouse models for preclinical study of ADCs combined with immunotherapy

Abstract Antibody-drug conjugates (ADCs) are effective anti-tumor therapies with high specificity, with little toxicity and side effects. Xenograft tumor models based on immunodeficient mice have been used for evaluating the human anti-tumor efficacy of ADCs. However, the antibody-mediated immune response of ADCs is not well evaluated in immunodeficient mice. To this end, we have developed BALB/c-hPD1 humanized mouse model to evaluate the efficacy of ADCs in combination with immunotherapy. Trastuzumab deruxtecan (T-DXd, DS-8201a) is a HER2-targeting ADC. In vitro efficacy studies showed that T-DXd effectively bound to the HER2-positive cell line SK-BR-3, resulting in strong endocytosis and induction of apoptosis. It has also been shown to kill HER2-negative MDA-MB-468 cells through a bystander effect both in vitro and in vivo. In vivo efficacy studies have demonstrated that ADCs can effectively inhibit human tumor cell growth in immunodeficient mice. In addition to human tumor cell lines, the efficacy of ADCs was also studied in BALB/c-hPD1mice engrafted with murine colon cancer cells model, and the results showed that T-DXd and Keytruda could significantly inhibit CT26-hHER2 tumor growth, with the tumor growth inhibition rates being 57% and 66%, respectively, Tumor growth inhibition rate of the combined treatment group was about 91%, which was superior compared to the efficacy of single drug treatment. The evaluation of Fc segment-mediated effect (such as ADCC, CDC and ADCP) of ADCs antibodies was carried out in humanized immune system mouse models, such as NCG-hIL15 and NCG-hSGM3. In summary, we have established a drug evaluation platform using various humanized mouse models to study the synergistic anti-tumor effects of ADCs combined with immune checkpoint blockade antibodies. Citation Format: Fang Zhu, Hongyan Sun, Qian Lu, Yuan Fang, Jianming Xu, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. Efficacy evaluation by novel humanized mouse models for preclinical study of ADCs combined with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3242.

Abstract 5071: Olverembatinib (HQP1351) enhances antitumor effects of immunotherapy in renal cell carcinoma (RCC)

Abstract In solid tumors, resistance to checkpoint inhibitors (CPIs) is frequently observed, partially due to upregulation of vascular endothelial growth factor A (VEGFA) and programmed death-ligand 1 (PD-L1). This culminates in an immunosuppressive tumor microenvironment and immune escape. Inhibitors against VEGF and the VEGF receptor (VEGFR) foster tumor vessel normalization and immunostimulatory reprogramming, in turn promoting treatment effects of immunotherapies. In recent years, TKIs, including axitinib, lenvatinib, and cabozantinib, plus immunotherapy have been approved to treat advanced RCC. Currently under clinical development for relapsed or refractory chronic myeloid leukemia and gastrointestinal tumor, olverembatinib (HQP1351) is a new-generation multikinase inhibitor with targets including VEGFR, fibroblast growth factor receptor (FGFR), SRC, BCR-ABL1, c-KIT, and platelet-derived growth factor receptor. The aim of this study was to assess whether olverembatinib combined with immunotherapy can promote inhibitory effects on RCC. In cell-free kinase assays, olverembatinib inhibited VEGFR1, -2, and -3 with IC50 values of 4.2, 6.1, and 4.1 nM, respectively. Compared to lenvatinib, olverembatinib had more potent antiproliferative effects on human umbilical vein endothelial cells. Olverembatinib also had antiproliferative activity in murine RCC lines RANCA and RAG, with IC50 values of 141 and 53 nM, respectively. When olverembatinib was coadministered with an anti-PD-1 antibody in a RANCA-derived syngeneic model, both agents exerted synergistic effects, with tumor growth inhibition rates reaching 60.6%. Mechanistically, olverembatinib influenced cancer cell proliferation directly by inhibiting phosphorylation of FGFR and downstream proteins. Increased cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 were observed, suggesting induction of apoptosis. Olverembatinib also influenced proliferation of vascular endothelial cells by inhibiting phosphorylation of VEGFR, SRC, and downstream proteins Akt and extracellular signal-regulated kinases. Olverembatinib also reduced expression of PD-L1 in RCC cells. In tumor-infiltrating lymphocyte assays, olverembatinib increased numbers of cytotoxic T cells (CTL, CD8+) and natural-killer cells (NK, CD3−/CD49B+) in RANCA tumor tissues. Combined with an anti-PD-1 antibody, olverembatinib increased CTLs, NK cells, dendritic cells (DCs, MHC-II+/CD11C+), and M1 macrophages (F4/80+/CD11B+/CD86+) in RANCA tumor tissues, indicating an immunoregulatory effect of olverembatinib. Taken together, these data suggest that combining olverembatinib with a CPI confers synergistic antitumor effects in an RCC cancer mouse model by targeting tumor growth, angiogenesis, and immune regulation. This novel combination may provide an alternative approach to enhance treatment effects with CPIs in renal cancers. Citation Format: Guangfeng Wang, Eric Liang, Ping Min, Huidan Yu, Bingxing Wu, Dajun Yang, Yifan Zhai. Olverembatinib (HQP1351) enhances antitumor effects of immunotherapy in renal cell carcinoma (RCC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5071.

Abstract 2847: Concurrent inactivation of PI3K and PLK1 is synergistic and overcomes acquired resistance to PI3K inhibitors in NOTCH1MUT HNSCC

Abstract Targeted therapies are limited for head and neck squamous cell carcinoma (HNSCC), as it is driven by mutations in tumor suppressors, including NOTCH1. We previously identified loss of function NOTCH1 mutations in HNSCC to be sensitive to phosphoinositide-3 kinase (PI3K) inhibitors through sustained Aurora kinase B levels. However, therapy resistance and modest responses are the leading causes of failure for targeted therapies. To address this pressing clinical need, we sought to identify drugs that would enhance the efficacy of PI3K inhibitors. We tested 5768 drugs (0-1µM) with diverse targets in NOTCH1 mutant (NOTCH1MUT - HN31, UMSCC22A, PCI-15B) HNSCC cell lines and copanlisib acquired resistant (CAR) HN31 clones. We determined drug efficacy using two metrics-area over the curve lethal dose (AOC_LD>0, cytotoxic) and area under the growth curve (AUC_GRI<0.9, cytostatic). These metrics are more robust than IC50 values as they use the normalized growth rate inhibition curve and avoid the confounding effect of the rate of cell division. Of 306 drug classes, 100 were effective with at least one drug being cytotoxic or cytostatic in at least one cell line. PLK inhibitors were the most effective class of drugs against NOTCH1MUT HNSCC cell lines (87% effective - 12 cytotoxic, 2 cytostatic, 2 ineffective). We further tested the PLK1-specific inhibitor onvansertib (0-100nM) combined with pan-PI3K inhibitor copanlisib (0-200nM) or dual inhibitor bimiralisib (0-1μM). We observed robust decreases in cell numbers at very low drug concentrations (50nM) with the combinations. We validated these results in vitro in NOTCH1MUT, NOTCH1WT, CAR NOTCH1MUT HNSCC models, and HEK293 by using independent approaches to test for apoptosis. A significant increase in cleaved PARP and cleaved caspase 3, and Annexin V/PI staining (>2x), was evident when PI3K and PLK1 were concurrently inactivated as compared to single agent treatment in HNSCC models. Furthermore, HEK293 cells were unaffected at these doses. We then investigated the role of PLK1 following PI3K inhibition in HNSCC models and found PLK1 protein levels to be downregulated. As PLK1 is downstream of Aurora kinases, we further determined the phospho-PLK1 (p-PLK1) levels in HNSCC models. Interestingly, p-PLK1 levels remained unaltered in NOTCH1WT and CAR NOTCH1MUT cells despite total protein level depletion. However, p-PLK1 levels drastically decreased in NOTCH1MUT HNSCC cells. This finding could explain the importance of PLK1 inactivation in addition to PI3K inhibition as a requirement for increased cell death in HNSCC models. We will further validate this combination in vivo to determine the effect of combined PI3K and PLK1 inhibition on tumor growth and survival. These novel findings may lead to the development of a better therapeutic approach for NOTCH1MUT HNSCC and for patients who develop acquired resistance to targeted therapies. Citation Format: Pooja A. Shah, Tuhina Mazumdar, Reid T. Powell, Li Shen, Jing Wang, Clifford C. Stephen, Mitchell J. Frederick, Faye M. Johnson. Concurrent inactivation of PI3K and PLK1 is synergistic and overcomes acquired resistance to PI3K inhibitors in NOTCH1MUT HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2847.

Effects of Vitamin E Derivative TMG on the Radiation Protector and Tumor Growth during Radiotherapy

Background: The purpose of this study is to evaluate the immunosuppressive and antioxidant effects of a novel radioprotective agent using the vitamin E derivative 2-(alpha-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (TMG) and its effect on tumors, and to study its usefulness.Materials and Methods: In this study, C57BL/6NCrSlc mice were divided into four groups (control, TMG, radiation therapy [RT], and RT+TMG), using 10 mice in each group. In the TMG and 2 Gy+TMG groups, 500 mg/kg TMG was administered. Two groups (2 Gy and 2 Gy+ TMG) among RT and RT+TMG groups were irradiated with 2 Gy in a single fraction, while the other two groups (6 Gy and 6 Gy+TMG) were irradiated locally with 6 Gy in three fractions.Results and Discussion: TMG positively affected CD4+ and CD8+ T lymphocytes. Tumor volumes and growth inhibition rates were compared. In order to evaluate how TMG administration affected tumor growth, Ehrlich cancer cells were injected into the thigh of mice, and the tumor volume and growth suppression rate were compared. Not only RT but also TMG alone inhibited tumor growth. If RT conducted to the mice with TMG, TMG could increase the number of leukocytes, primarily that of lymphocytes. TMG also inhibited tumor growth in addition to RT. Tumor growth was significantly inhibited in the 6 Gy+TMG group.Conclusion: In conclusion, TMG exerted an immunopotentiating effect mainly by increasing the white blood cell numbers including that of lymphocytes. In addition to RT, TMG also inhibited tumor growth. Therefore, TMG is considered to be a useful radioprotective agent in radiotherapy without tumor growth induction.

Transcriptomic analysis of the effect of deferoxamine exposure on the growth, photosynthetic activity and iron transfer of Microcystis aeruginosa

Deferoxamine (DFB) is a trihydroxamic acid siderophore that chelates with iron (Fe) to form iron-siderophore complexes. The existence of siderophores in nature changes the form of iron and affects the absorption and utilization of iron by organisms. However, the relationship between siderophores and the growth of Cyanobacteria is largely unknown. In this study, the cellular and transcriptomic responses to the addition of DFB were investigated. A high concentration of DFB (12 mg/L) significantly inhibited the growth of Cyanobacteria cells, reduced photosynthetic activity, and induced the production of peroxidase, with the highest inhibition rate of algal growth of 74.82%. These indexes were also affected for the low (3 mg/L) and medium concentration (6 mg/L) groups, but this difference is closely related to the growth stage of Cyanobacteria cells. This may be due to competition between the cell-associated iron-binding part/system and the extracellular Fe (Ⅲ)-DFB ligand. Transcriptome results showed that most of the genes involved in iron uptake and transport were down-regulated, and only the fur gene encoding the iron uptake regulator protein was significantly up-regulated. Most genes related to photosynthesis, glycolysis, and fatty acid metabolism were also down-regulated, while the obvious up-regulation of a few genes may be a complex regulation in response to the down-regulation of most genes. These findings will provide important insights into the effects of siderophores on iron bioavailability in algae.

Efficient electrochemical oxidation of cephalosporin antibiotics by a highly active cerium doped PbO2 anode: Parameters optimization, kinetics and degradation pathways

Removing cephalosporin contaminants from aquatic environments is of great concern because of the hazards they pose to the environment and human health. In this study, a Ce-PbO2 anode with a SnO2-Sb intermediate layer was fabricated using an electrochemical deposition method and used for the electrocatalytic oxidation of cephalosporin antibiotics. Scanning electron microscopy (SEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) confirmed the successful preparation of the Ce-PbO2 anode and revealed that Ce doping resulted in a highly stable structure and crystallinity of the PbO2 crystals. Moreover, the Ce-PbO2 anode exhibited excellent electrochemical performance with higher oxygen evolution potential (1.56 V), larger electroactive surface area (1.104 C cm−2), smaller corrosion current density (1.796 × 10−4 A cm−2), and higher hydroxyl radical (•OH) yield (8.64 mM min−1 m−2) compared with the intermediate layer and PbO2 anode. The effects of major operating parameters, including current density (20–80 mA cm−2), electrolyte concentration (0.050–0.125 M), initial concentration of target compounds (5–75 mg L−1), and pH (2−10) on the removal efficiencies of cefradine (RAD), and cephalothin (CEP) at the Ce-PbO2 electrode were investigated. The research results illustrated that when electrolysis was carried out for 90 min at the optimal current density of 40 mA cm−2, an initial concentration of 50 mg L−1, and an electrolyte concentration of 0.10 M, the removal efficiency of RAD and CEP were higher than 99%. Additionally, the degradation process followed pseudo-first-order kinetics. The total organic carbon (TOC) removal efficiency of RAD and CEP was 60.6% and 68.2% after 240 min, respectively, under the optimal conditions. The energy consumption for RAD and CEP degradation was 1.74 and 3.04 kWh g−1 TOC, respectively. In addition, the Ce-PbO2 electrode exhibited good stability over nine consecutive cycles. Using UPLC-MS, the intermediate products of RAD and CEP were confirmed, and the degradation intermediates were completely oxidized to CO2, H2O, and inorganic ions (SO42-, NO3-). During electrolysis, the intermediate toxicity was studied using Escherichia coli growth inhibition tests, and the growth inhibition rate was calculated. Considering these results, the Ce-PbO2 anode could be utilized to treat wastewater containing cephalosporin antibiotics with high efficiency.

Design, synthesis, and biological evaluation of 6-(imidazo[1,2-a] pyridin-6-yl) quinazolin-4(3H)-one derivatives as potent anticancer agents by dual targeting Aurora kinase and ROR1

ROR1 and Aurora kinase were overexpressed in various cancers and essential for cell proliferation, survive and metastasis. Pharmaceutical inhibition of ROR1 and Aurora kinase abrogated the activation of downstream signaling and induced cancer cell apoptosis. Hence, ROR1 and Aurora kinase considered as attractive therapeutic targets for the development of anticancer drugs. In the present work, three series of novel 6-(imidazo[1,2-a] pyridin-6-yl)-quinazolin-4(3H)-one derivatives were designed and synthesized via bioisosterism and scaffold-hopping strategies guided by FLF-13, an Aurora kinase inhibitor we discovered earlier. Most of compounds in series 2 and series 3 showed submicromolar to nanomolar inhibitory activity against multiple cancer cell lines. More importantly, compounds 12d and 12f in series 3 showed nanomolar inhibitory activity against all test cancer cells. The most promising compound 12d exhibited potent inhibitory activity against Aurora A and Aurora B with IC50 values of 84.41 nM and 14.09 nM, respectively. Accordingly, compounds 12d induced G2/M phase cell cycle arrest at 24 h and polyploidy at 48 h. It's worth noting that 12d also displayed inhibitory activity against ROR1 and induce cell apoptosis. Furthermore, 12d could significantly inhibit the tumor growth in SH-SY5Y xenograft model with tumor growth inhibitory rate (IR) up to 46.31 % at 10 mg/kg and 52.66 % at 20 mg/kg. Overall, our data suggested that 12d might serve as a promising candidate for the development of therapeutic agents for cancers with aberrant expression of ROR1 and Aurora kinases by simultaneously targeting ROR1 and Aurora kinase.

Myrochromanol analogues and trichothecene derivatives from the shellfish-derived fungus Albifimbriaverrucaria

Eight myrochromanol analogues, including three pairs of epimers at C-2 with the myrochromanol scaffold and two examples of myrochromanol with sugar moiety linked at C-4, together with twelve trichothecene derivatives were isolated from the cultures of a shellfish-derived fungus Albifimbria verrucaria CD1-4. Among them, eight compounds named 2-epi-myrochromanol, ent-myrochromanol B, 4-epi-myrochromanol B, 2-epi-myrochromanol A, myrochromanosides A and B, 6′,7′-erythro-(2′E,4′Z)-trichoverrol B, 3R,8S-dihyroxyroridin H were previously undescribed fungal metabolites. Their planar structures and relative configurations were established by 1D and 2D NMR, and HR-MS data analysis, and their absolute configurations were determined using the modified Mosher's method and electronic circular dichrosim calculations. Almost all isolates were evaluated for growth rate inhibition of three marine harmful microalgae Chattonella marina, Heterosigma akashiwo, and Prorocentrum donghaiense, and lethal activity to one marine zooplankton, Artemia salina. Myrochromanosides A and B exhibited obvious inhibitory against three tested microalgae with IC50 values in the range of 9.2–108.9 μM. 8α-Hydroxyroridin H, roridin A and verrucarin A exhibited significant inhibition against P. donghaiense with IC50 values of 6.1, 5.8, and 6.0 μM and toxicity against brine shrimp larvae with LC50 values of 1.4, 2.8, and 0.26 μM, respectively.

Multicomponent synthesis of new 5‐thiourea‐4‐aza‐2,3 didehydropodophyllotoxins as potent cytotoxic agents

AbstractIn this report, we have described the concise elaboration of 12 novel 5‐thiourea‐4‐aza‐2,3‐didehydropodophyllotoxin‐derivatives by the unconventional multicomponent reaction was based on a catalyst‐free and atom‐economical method. The synthetic approach features methodology using sulfur element in thiourea synthesis. Cytotoxicity of compounds was evaluated for their inhibition rate of cell growth against two human tumor cell lines (MCF‐7 and Hep‐G2). Some new 5‐thiourea‐4‐aza‐2,3‐didehydropodophyllotoxin such as 15a, 15d, 15l exhibit very good cytotoxicity and much more stronger than their precursor 5 amino‐4‐aza‐2,3‐didehydropodophyllotoxin. The number and chemical characters of the Cα substituents of the thiourea group may have a decisive role in the activity of the molecules. Docking studies of the tested molecules proved that compound 15a, 15d, 15l exhibit high binding affinity toward human topoisomerase II beta in compare to the reference inhibitor, etoposide. Further dock pose analysis sugessted these molecules as potential inhibitors‐based on their key interaction with residues within binding site of the protein.

Local delivery of artesunate dimer liposomes incorporated injectable hydrogel for H2O2 and pH-independent chemodynamic therapy

Chemodynamic therapy (CDT) has emerged as a powerful tumor treatment option by inducing the imbalance of redox homeostasis in cancer cells. Nevertheless, the therapeutic outcomes were greatly limited because of insufficient endogenous H2O2 and upregulated cellular antioxidant defense in the tumor microenvironment (TME). Herein, a liposome-incorporated in-situ alginate hydrogel locoregional treatment strategy was developed, which involves using hemin-loaded artesunate dimer liposomes (HAD-LP) as redox-triggered self-amplified C-center free radical nanogenerator to enhance CDT. First, HAD-LP based on artesunate dimer glycerophosphocholine (ART-GPC) was prepared by a thin film method. Their spherical structure was manifested by dynamic light scattering (DLS) and transmission electron microscope (TEM). The generation of C-center free radicals from HAD-LP was carefully evaluated by using methylene blue (MB) degradation method. The results suggested that the hemin was reduced to heme under the action of glutathione (GSH), which could catalyze the breakage of endoperoxide of ART-GPC derived dihydroartemisinin (DHA) to generate toxic C-centered free radicals in a H2O2 and pH-independent manner. Moreover, the change of intracellular GSH and free radical level was monitored through ultraviolet spectroscopy and confocal laser scanning microscope (CLSM). It was revealed that the hemin reduction induced GSH depletion and elevated free radical level, disrupting cellular redox homeostasis. After co-incubation with MDA-MB-231 or 4 T1 cells, HAD-LP was found to be highly cytotoxic. In order to prolong retention and improve antitumor efficacy, HAD-LP was mixed with alginate and injected intratumorally into 4 T1 tumor bearing mice. The injected HAD-LP and alginate mixture formed in-situ hydrogel and achieved best antitumor effect with the growth inhibition rate of 72.6%. Together, the hemin-loaded artesunate dimer liposome-incorporated alginate hydrogel possessed effective antitumor activity through redox-triggered C-center free radical generation induced apoptosis in a H2O2 and pH-independent manner, which might be a promising candidate in the application of chemodynamic anti-tumor therapy.

Evaluation of In Vitro and In Vivo Antifungal Activity of Green Synthesized Silver Nanoparticles against Early Blight in Tomato

Silver nanoparticles have gained considerable interest in recent decades due to their antimicrobial activity and are used in water disinfection, wound healing, food packaging, and plant protection. This study tested the potential of silver nanoparticles synthesized using the neem (Azadirachta indica) leaf extract against Alternaria solani causes early blight disease in tomato plants. The pathogen was isolated from infected tomato plants and identified using morphological and molecular features. The results showed significant variation among isolates. Isolates, Shk-1 and Ksr-1 were highly pathogenic, causing up to 80% disease incidence. The potential of silver nanoparticles against each isolate was determined using different concentrations of silver nanoparticles. During in vitro and in vivo experiments, the growth inhibition rate of the pathogen was 70–100% at 50 ppm. Lower concentrations of silver nanoparticles (5 and 10 ppm) increased phenolics, PO, PPO, and PAL production by more than 50% as compared to the untreated control. These defensive mechanisms clearly demonstrate the fungicidal potential of AgNPs and recommend their utilization in different crop protection programs.

Natural rubber latex films with effective growth inhibition against S. aureus via surface conjugated gentamicin

Hospital-associated infections and related complications are of extreme concern in the healthcare sector since biofilms generated over material surfaces not only create turbulence in the healthcare practices followed but also ruin the device performance, and increased medication, leading to significant chances of drug resistance. Natural rubber latex (NRL) being the first choice for the manufacture of several conventional biomedical devices, it is essential to ensure the surfaces of the same are inherently inactive against most microorganisms. This study presents NRL film surface conjugated with a well-known antibiotic, gentamicin through an amide linkage to generate antibacterial activity to the surface with a significant growth inhibition rate, especially against Staphylococcus aureus. The NRL films were surface-oxidized under controlled acidic conditions to generate carboxyl groups exploring the unsaturation of the base monomer unit. The carboxyl group reacts with the amine groups of gentamicin facilitating its surface conjugation. The surface anchoring was authenticated by FTIR-ATR complimented further by contact angle measurement as a function of hydrophilicity and elemental analysis by EDX spectroscopy. The antibacterial efficacy of modified NRL films was evaluated using antibacterial drop test and the results indicated a substantial growth inhibition rate (>60%) against Pseudomonas aeruginosa and Staphylococcus aureus. The study could be further optimized and proposed as a viable route for the conjugation of active molecules over inert polymer molecules.

Enzymatic, Antioxidant, and Antimicrobial Activities of Bioactive Compounds from Avocado (Persea americana L.) Seeds

The aim of this research was to identify and quantify biologically active compounds from avocado (Persea americana L.) seeds (AS) utilizing different techniques with the use of ultrasound (US), ethanol (EtOH), and supercritical carbon dioxide (scCO2) for possible applications in (bio)medicine, pharmaceutical, cosmetic, or other relevant industries. Initially, a study of the process efficiency (η) was carried out, which revealed yields in the range of 2.96-12.11 wt%. The sample obtained using scCO2 was found to be the richest in total phenols (TPC) and total proteins (PC), while the sample obtained with the use of EtOH resulted in the highest content of proanthocyanidins (PAC). Phytochemical screening of AS samples, quantified by the HPLC method, indicated the presence of 14 specific phenolic compounds. In addition, the activity of the selected enzymes (cellulase, lipase, peroxidase, polyphenol oxidase, protease, transglutaminase, and superoxide dismutase) was quantified for the first time in the samples from AS. Using DPPH radical scavenging activity, the highest antioxidant potential (67.49%) was detected in the sample obtained with EtOH. The antimicrobial activity was studied using disc diffusion method against 15 microorganisms. Additionally, for the first time, the antimicrobial effectiveness of AS extract was quantified by determination of microbial growth-inhibition rates (MGIRs) at different concentrations of AS extract against three strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas fluorescens) bacteria, three strains of Gram-positive (Bacillus cereus, Staphylococcus aureus, and Streptococcus pyogenes) bacteria, and fungi (Candida albicans). MGIRs and minimal inhibitory concentration (MIC90) values were determined after 8 and 24 h of incubation, thus enabling the screening of antimicrobial efficacy for possible further applications of AS extracts as antimicrobial agents in (bio)medicine, pharmaceutical, cosmetic, or other industries. For example, the lowest MIC90 value was determined for B. cereus after 8 h of incubation in the case of UE and SFE extracts (70 μg/mL), indicating an outstanding result and the potential of AS extracts, as the MIC values for B. cereus have not been investigated so far.

Bacillus cereus B8W8 an effective bacterial antagonist against major postharvest fungal pathogens of fruit

The antifungal activity of Bacillus cereus (B8W8) was investigated against major post-harvest fungal pathogens affecting citrus and apples fruits namely Penicillium digitatum, Penicillium italicum, Geotrichum citri-aurantii, Penicillium expansum, Botrytis cinerea, Monilinia laxa and Monilinia fructigena. The in vitro dual culture of B8W8 and fungal pathogens showed high inhibition rates of the mycelial growth of Monilinia laxa and Penicillium digitatum (71.4% and 72.5% respectively). Subsequently, the volatile compounds (VOCs) have a high antagonistic effect against Monilinia laxa (86.4%) and Monilinia fructigena (73.4%). In addition, the bacterial-free cell filtrate of B8W8 had a significantly important effect on the inhibition of spore’s germination and mycelial growth of Penicillium expansum, Botrytis cinerea, Penicillium italicum and Geotrichum citri-aurantii. The in vivo bioassays revealed a significant impact of B8W8 on fruit decays either by reducing the disease severity of causative agents or by completely preventing their occurrence, particularly brown rot of apple caused by M. laxa and green mold of citrus caused by P. digitatum (DS = 0%). However, the severity of sour rot of citrus caused by G. citri-aurantii was only reduced by 50%. The semi-commercial trials showed a high potential of B. cereus B8W8 to reduce the incidence of gray mold of apple and green mold of citrus to very low values during at storage. In addition, the biological treatment with B8W8 was shown generally more effective than imazalil, the chemical treatment reference. Furthermore, the rapid colonization of fruit wounds by B8W8 could promote its antagonistic ability. Moreover, biochemical tests showed the ability of B8W8 to produce protease, amylase, and hydrocyanic acid (HCN). Interestingly, the molecular analysis demonstrated the ability of this bacterium to produce the fengycin. The secretion of this lipopeptide and lytic enzymes is more likely the major mechanism of action involved in the biocontrol activity of this bacterium. Results indicated that quality parameters of fruit, when challenged post-harvest pathogens and treated with B8W8, were mostly very close to those of the untreated control, suggesting that B8W8 did not affect the fruit quality. Accordingly, it was concluded that B. cereus strain B8W8 could be an effective biological agent to control the main fungal pathogens of apples and citrus fruits during post-harvest period.

Synergistic Effect of Lenvatinib and Chemotherapy in Hepatocellular Carcinoma Using Preclinical Models.

The current study aimed to evaluate the synergistic efficacy of lenvatinib and FOLFOX (infusional fluorouracil (FU), folinic acid, and oxaliplatin) in hepatocellular carcinoma (HCC) using patient-derived xenograft (PDX) and PDX-derived organotypic spheroid (XDOTS) models in vivo and in vitro. PDX and matched XDOTS models originating from three patients with HCC were established. All models were divided into four groups and treated with drugs alone or in combination. Tumor growth in the PDX models was measured and recorded, and angiogenesis and phosphorylation of the vascular endothelial growth factor receptor (VEGFR2), rearranged during transfection (RET), and extracellular signal-regulated kinase (ERK) were detected using immunohistochemistry and Western blot assays. The proliferative ability of XDOTS was evaluated through active staining and immunofluorescence staining, and the effect of the combined medication was evaluated using the Celltiter-Glo luminescent cell viability assay. Three PDX models with genetic characteristics similar to those of the original tumors were successfully established. Combining lenvatinib with FOLFOX led to a higher tumor growth inhibition rate than individual therapies (P < 0.01). Immunohistochemical analysis demonstrated that the combined treatment significantly inhibited the proliferation and angiogenesis of PDX tissues (P < 0.05), and Western blot analysis showed that the combined treatment significantly inhibited the phosphorylation of VEGFR2, RET, and ERK compared with single-agent treatment. Additionally, all three matched XDOTS models were successfully cultured with satisfactory activity and proliferation, and the combined therapies led to better suppression of XDOTS growth compared with individual therapy (P < 0.05). Lenvatinib combined with FOLFOX had a synergistic antitumor effect in HCC PDX and XDOTS models by inhibiting the phosphorylation of VEGFR, RET, and ERK.

Inhibitory effect of three strains of biocontrol microbes on pathogens causing rhizome rot of Polygonatum cyrtonema

Rhizome rot is one of the main disease in the cultivation of Polygonatum cyrtonema, and it is also a global disease which seriously occurs on the perennial medicinal plants such as Panax notoginseng and P. ginseng. There is no effective control method at present. To identify the effects of three biocontrol microbes(Penicillium oxalicum QZ8, Trichoderma asperellum QZ2, and Brevibacillus amyloliquefaciens WK1) on the pathogens causing rhizome rot of P. cyrtonema, this study verified six suspected pathogens for their pathogenicity on P. cyrtonema. The result showed that Fusarium sp. HJ4, Colletotrichum sp. HJ4-1, and Phomopsis sp. HJ15 were the pathogens of rhizome rot of P. cyrtonema, and it was found for the first time that Phomopsis sp. could cause rhizome rot P. cyrtonema. Furthermore, the inhibitory effects of biocontrol microbes and their secondary metabolites on three pathogens were determined by confrontation culture. The results showed that the three tested biocontrol microbes significantly inhibited the growth of three pathogens. Moreover, the secondary metabolites of T. asperellum QZ2 and B. amyloliquefaciens WK1 showed significant inhibition against the three pathogens(P&lt;0.05), and the effect of B. amyloliquefaciens WK1 sterile filtrate was significantly higher than that of high tempe-rature sterilized filtrate(P&lt;0.05). B. amyloliquefaciens WK1 produced antibacterial metabolites to inhibit the growth of pathogens, and the growth inhibition rate of its sterile filtrate against three pathogens ranged from 87.84% to 93.14%. T. asperellum QZ2 inhibited the growth of pathogens through competition and antagonism, and P. oxalicum QZ8 exerted the inhibitory effect through competition. The research provides new ideas for the prevention and treatment of rhizome rot of P. cyrtonema and provides a basis for the di-sease control in other crops.