Predicting the mechanism of action of YQYYJD prescription in the treatment of non-small cell lung cancer using transcriptomics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The efficacy of the herbal formula Yiqi Yangyin Jiedu (YQYYJD) in the treatment of advanced lung cancer has been reported in clinical trials. However, the key anti-lung cancer herbs and molecular mechanisms underlying its inhibition of lung cancer are not well-understood. AIM OF THE STUDY: To identify the key anti-lung cancer herbs in the YQYYJD formula and investigate their therapeutic effect and potential mechanism of action in non-small cell lung cancer (NSCLC) using transcriptomics and bioinformatics techniques. MATERIALS AND METHODS: A mouse Lewis lung carcinoma (LLC) subcutaneous inhibitory tumor model was established with 6 mice in each group. Mice were treated with the YQYYJD split formula: Yiqi Formula (YQ), Yangyin Formula (YY), and Ruanjian Jiedu Formula (RJJD) for 14 days. The tumor volume and mouse weight were recorded, and the status of tumor occurrence was further observed by taking photos. The tumor was stained with hematoxylin-eosin to observe its histopathological changes. Immunohistochemistry was used to detect the expression of the proliferation marker Ki67 and the apoptotic marker Caspase-3 in tumor tissues. Flow cytometry was used to detect the number of CD4+ and CD8+ T cells and cytokines interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in the spleen and tumor tissues. The differential genes of key drugs against tumors were obtained by transcriptome sequencing of tumors. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analyses were performed on differential genes to obtain pathways and biological processes where targets were aggregated. TIMER2.0 and TISIDB databases were used to evaluate the impact of drugs on immune cell infiltration and immune-related genes. The binding activity of the key targets and compounds was verified by molecular docking. RESULTS: YQ, YY, and RJJD inhibited the growth of subcutaneous transplanted tumors in LLC mice to varying degrees and achieved antitumor effects by inhibiting the expression of tumor cell proliferation, apoptosis, and metastasis-related proteins. Among the three disassembled prescriptions, YQ better inhibited the growth of subcutaneous transplanted tumors in LLC mice, significantly promoted tumor necrosis, significantly increased the expression of Caspase-3 protein in tumor tissue, and significantly decreased the expression of Ki-67 (P < 0.05), thereby increasing the infiltration of CD8+ T cells. YQ significantly increased the expression of CD4+ and CD8+ T cells in tumor and splenic tissues of tumor-bearing mice and up-regulated the expression of IL-2 and IFN-γ. Transcriptome sequencing and bioinformatics results showed that after YQ intervention, differentially expressed genes were enriched in more than one tumor-related pathway and multiple immune regulation-related biological functions. There were 12 key immune-related target genes. CONCLUSION: YQ was the key disassembled prescription of YQYYJD, exerting significant antitumor effects and immune regulation effects on NSCLC. It may have relieved T cell exhaustion and regulated the immune microenvironment to exert antitumor effects by changing lung cancer-related targets, pathways, and biological processes.

Co-encapsulation of fisetin and cisplatin into liposomes: Stability considerations and in vivo efficacy on lung cancer animal model.

Lung cancer is a highly vascularized tumor for which a combination between an antitumor agent, cisplatin, and an antiangiogenic molecule, fisetin, appears a promising therapeutic approach. In order to deliver both chemotherapies within the tumor, to enhance fisetin solubility and decrease cisplatin toxicity, an encapsulation of both drugs into liposomes was developed. Purification and freeze-drying protocols were optimized to improve both the encapsulation and liposome storage. The cytotoxicity of the encapsulated chemotherapies was evaluated on Lewis lung carcinoma (3LL) cell lines. The antitumor effect of the combination was evaluated in vivo on an ectopic mouse model of Lewis Lung carcinoma. The results showed that fisetin and cisplatin co-loaded liposomes were successfully prepared. Freeze-drying allowed a 30 days storage limiting the release of both drugs. The combination index between liposomal fisetin and liposomal cisplatin on 3LL cell line after 24 h of exposure showed a clear synergism: CI = 0.7 for the co loaded liposomes and CI = 0.9 for the mixture of cisplatin loaded and fisetin loaded liposomes. The co-encapsulating formulation showed in vivo efficacy against an ectopic murine model of Lewis Lung carcinoma with a probable reduction in the toxicity of cisplatin through co-encapsulation with fisetin.

Opaganib (ABC294640) Induces Immunogenic Tumor Cell Death and Enhances Checkpoint Antibody Therapy.

Antibody-based cancer drugs that target the checkpoint proteins CTLA-4, PD-1 and PD-L1 provide marked improvement in some patients with deadly diseases such as lung cancer and melanoma. However, most patients are either unresponsive or relapse following an initial response, underscoring the need for further improvement in immunotherapy. Certain drugs induce immunogenic cell death (ICD) in tumor cells in which the dying cells promote immunologic responses in the host that may enhance the in vivo activity of checkpoint antibodies. Sphingolipid metabolism is a key pathway in cancer biology, in which ceramides and sphingosine 1-phosphate (S1P) regulate tumor cell death, proliferation and drug resistance, as well as host inflammation and immunity. In particular, sphingosine kinases are key sites for manipulation of the ceramide/S1P balance that regulates tumor cell proliferation and sensitivity to radiation and chemotherapy. We and others have demonstrated that inhibition of sphingosine kinase-2 by the small-molecule investigational drug opaganib (formerly ABC294640) kills tumor cells and increases their sensitivities to other drugs and radiation. Because sphingolipids have been shown to regulate ICD, opaganib may induce ICD and improve the efficacy of checkpoint antibodies for cancer therapy. This was demonstrated by showing that in vitro treatment with opaganib increases the surface expression of the ICD marker calreticulin on a variety of tumor cell types. In vivo confirmation was achieved using the gold standard immunization assay in which B16 melanoma, Lewis lung carcinoma (LLC) or Neuro-2a neuroblastoma cells were treated with opaganib in vitro and then injected subcutaneously into syngeneic mice, followed by implantation of untreated tumor cells 7 days later. In all cases, immunization with opaganib-treated cells strongly suppressed the growth of subsequently injected tumor cells. Interestingly, opaganib treatment induced crossover immunity in that opaganib-treated B16 cells suppressed the growth of both untreated B16 and LLC cells and opaganib-treated LLC cells inhibited the growth of both untreated LLC and B16 cells. Next, the effects of opaganib in combination with a checkpoint antibody on tumor growth in vivo were assessed. Opaganib and anti-PD-1 antibody each slowed the growth of B16 tumors and improved mouse survival, while the combination of opaganib plus anti-PD-1 strongly suppressed tumor growth and improved survival (p < 0.0001). Individually, opaganib and anti-CTLA-4 antibody had modest effects on the growth of LLC tumors and mouse survival, whereas the combination of opaganib with anti-CTLA-4 substantially inhibited tumor growth and increased survival (p < 0.001). Finally, the survival of mice bearing B16 tumors was only marginally improved by opaganib or anti-PD-L1 antibody alone but was nearly doubled by the drugs in combination (p < 0.005). Overall, these studies demonstrate the ability of opaganib to induce ICD in tumor cells, which improves the antitumor activity of checkpoint antibodies.

The triple-drug combination DBDx enhances the antitumor efficacy of PD-1 antibody associated with Treg modulation.

OBJECTIVE: This study investigated the antitumor efficacy of programmed cell death protein-1 (PD-1) antibody and DBDx, a triple-drug combination of dipyridamole, bestatin, and dexamethasone, and their related immunomodulation. MATERIALS AND METHODS: Mouse melanoma B16, mouse Lewis lung carcinoma, and mouse breast carcinoma 4T1 were used for evaluating the in vivo therapeutic efficacy of DBDx, PD-1 antibody, and their combination. The peripheral blood and tumor tissues of 4T1 tumor-bearing mice were collected to analyze regulatory T cells and measured using flow cytometry. RESULTS: The combination of PD-1 antibody and DBDx enhanced the therapeutic efficacy against B16 melanoma. The suppression of tumor growth by PD-1 antibody and DBDx was more significant than that by anti-PD-1 monotherapy. The tumor growth inhibition rates of PD-1 antibody, DBDx, and their combination were 54.0%, 72.4%, and 83.1%, respectively, suggesting a synergistic effect as determined by the coefficient of drug interaction. No significant changes were found in the body weights in all the above groups, indicating that the treated mice tolerated the applied drug doses. Similarly, enhanced therapeutic efficacy of the PD-1 antibody and DBDx combination was observed in murine Lewis lung carcinoma and 4T1 breast cancer models. In 4T1 breast cancer-bearing mice, the immunotherapy-related changes in lymphocytes in peripheral blood and tumor microenvironment were evaluated with flow cytometry. Compared with anti-PD-1 monotherapy, peripheral blood and tumor-infiltrating lymphocytes were found a lower ratio of regulatory T cell (Treg) subset cells and a higher ratio of CD8+/Treg cells. CONCLUSIONS: The combination of PD-1 antibody and DBDx could achieve enhanced therapeutic antitumor efficacy than anti-PD-1 monotherapy, suggesting potential for using the triple-drug combination DBDx in cancer immunotherapy.

Effect of Moxibustion Combined With Cisplatin on Tumor Microenvironment Hypoxia and Vascular Normalization in Lewis Lung Cancer Mice.

PURPOSE: This study was developed to evaluate the effects of moxibustion on tumor microenvironmental hypoxia in a murine model of Lewis lung carcinoma (LLC). METHODS: Twenty-four tumor-bearing mice were randomized into tumor group (T), tumor + cisplatin group (TC), tumor + moxibustion group (TM), and tumor + cisplatin + moxibustion group (TMC) (n = 6/group). Six age-matched C57BL/6 mice were employed as control group (Ctrl). A tumor model was established by implanting LLC cells into the right flank of each mouse. Animals in the TM group received moxibustion treatment at the ST36 (bilateral) and GV4 acupoints on the day of visible tumor formation. Moxibustion treatment was performed every other day for a total of 7 sessions. Animals in the TC group were intraperitoneally injected with cisplatin (3 mg/kg) on day 3 after visible tumor formation, and this treatment was performed every 3 days for 4 times. Animals in the TMC group underwent combined moxibustion and chemotherapy treatment, following the same conditions as outlined above. Following treatment, the concentrations of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), CD31, and Ki67 were measured using ELISA, Western blot, and immunohistochemical staining. RESULTS: Compared to the tumor group, treatment in the TM, TC, and TCM groups resulted in varying reductions in tumor growth (P < .001 or P < .05), while tumor microenvironmental hypoxia was alleviated as evidenced by the downregulation of HIF-1α, VEGFA, and CD31(P < .001-P < .05). CONCLUSION: Our results suggest that a combined approach of moxibustion and cisplatin can alleviate intratumoral hypoxia, promote vascular normalization, and slow the growth of LLC tumors in mice.

Photodynamic therapy of lung cancer with photosensitizers based on polycationic derivatives of synthetic bacteriochlorin (experimental study).

BACKGROUND: One of the tasks of anticancer photodynamic therapy is increasing the efficacy of treatment of cancer nodes with large (clinically relevant) sizes using near-infrared photosensitizers (PS). METHODS: The anticancer efficacy and mechanisms of the photodynamic action of PS based on polycationic derivatives of synthetic bacteriochlorin against Lewis lung carcinoma were studied in vitro and in vivo. RESULTS: It was found that studied PS have high phototoxicity against Lewis lung carcinoma cells: the IC50 values were about 0.8 µM for tetracationic PS and 0.5 µM for octacationic PS. In vivo studies have shown that these PS provide effective inhibition of the tumor growth with an increase in the lifespan of mice in the group by more than 130%, and more than 50% survival of mice in the group. CONCLUSIONS: Photosensitizers based on polycationic derivatives of synthetic bacteriochlorin have high photodynamic efficacy caused by the induction of necrosis and apoptosis of cancer cells, including cancer stem cells, and a sharp decrease of mitotic and proliferative activity. Studied polycationic photosensitizers are much more effective at destroying cancer stem cells and newly formed cancer vessels in comparison with anionic photosensitizers, and ensure the cessation of tumor blood flow without hemorrhages and thrombosis.

Effects of S-pindolol in mouse pancreatic and lung cancer cachexia models.

BACKGROUND: It is known that S-pindolol attenuates muscle loss in animal models of cancer cachexia and sarcopenia. In cancer cachexia, it also significantly reduced mortality and improved cardiac function, which is strongly compromised in cachectic animals. METHODS: Here, we tested 3 mg/kg/day of S-pindolol in two murine cancer cachexia models: pancreatic cancer cachexia (KPC) and Lewis lung carcinoma (LLC). RESULTS: Treatment of mice with 3 mg/kg/day of S-pindolol in KPC or LLC cancer cachexia models significantly attenuated the loss of body weight, including lean mass and muscle weights, leading to improved grip strength compared with placebo-treated mice. In the KPC model, treated mice lost less than half of the total weight lost by placebo (-0.9 ± 1.0 vs. -2.2 ± 1.4 g for S-pindolol and placebo, respectively, P < 0.05) and around a third of the lean mass lost by tumour-bearing controls (-0.4 ± 1.0 vs. -1.5 ± 1.5 g for S-pindolol and placebo, respectively, P < 0.05), whereas loss of fat mass was similar. In the LLC model, the gastrocnemius weight was higher in sham (108 ± 16 mg) and S-pindolol tumour-bearing (94 ± 15 mg) mice than that in placebo (83 ± 12 mg), whereas the soleus weight was only significantly higher in the S-pindolol-treated group (7.9 ± 1.7 mg) than that in placebo (6.5 ± 0.9). Grip strength was significantly improved by S-pindolol treatment (110.8 ± 16.2 vs. 93.9 ± 17.1 g for S-pindolol and placebo, respectively). A higher grip strength was observed in all groups; whereas S-pindolol-treated mice improved by 32.7 ± 18.5 g, tumour-bearing mice only show minimal improvements (7.3 ± 19.4 g, P < 0.01). CONCLUSIONS: S-pindolol is an important candidate for clinical development in the treatment of cancer cachexia that strongly attenuates loss of body weight and lean body mass. This was also seen in the weight of individual muscles and resulted in higher grip strength.

Eicosapentaenoic Acid-Enriched Phospholipids Alleviate Skeletal Muscle Atrophy in Lewis Lung Carcinoma Mouse Model.

SCOPE: Skeletal muscle atrophy is a critical feature of cancer-associated cachexia (CAC) and it is responsible for poor quality of life and high mortality in cancer patients. The previous study demonstrates that eicosapentaenoic acid-enriched phospholipids (EPA-PL) prevent body weight loss in a mouse model of CAC. However, the role of EPA-PL on cancer-induced skeletal muscle atrophy remains unclear. METHODS AND RESULTS: In the present study, a Lewis lung carcinoma (LLC) mouse model is established, then the effect and underlying mechanism of EPA-PL on skeletal muscle atrophy in LLC-bearing mice are investigated. The results reveal that EPA-PL treatment significantly attenuates skeletal muscle atrophy in LLC-bearing mice, as evidenced by suppressing the reductions of skeletal muscle mass, myofiber cross-sectional area, and grip strength. Besides, the study finds that EPA-PL alleviated cancer-induced skeletal muscle atrophy via balancing muscle protein degradation and synthesis, inhibiting type I oxidative muscle fibers atrophy, and promoting mitochondrial function. Furthermore, the results also indicate that EPA-PL may counteract skeletal muscle atrophy in LLC mouse model via a sirtuin 1-dependent mechanism. CONCLUSION: These findings provide evidence that EPA-PL may be beneficial as a nutritional supplement for prevention and treatment of cancer-induced skeletal muscle atrophy.

EXT418, a novel long-acting ghrelin, mitigates Lewis lung carcinoma induced cachexia in mice.

BACKGROUND: Ghrelin is a potential therapy for cachexia due to its orexigenic properties and anabolic effects on muscle and fat. However, its clinical use is limited by the short half-life of active (acylated) ghrelin (~11 min in humans). EXT418 is a novel long-acting, constitutively active ghrelin analog created by covalently linking it to a vitamin D derivative. Here, we evaluated the effects and mechanisms of action of EXT418 on Lewis lung carcinoma (LLC)-induced cachexia in mice. METHODS: Male C57BL/6J mice (5- to 7-month-old) were implanted with 1 × 106 heat-killed (HK) or live LLC cells. When the tumour was palpable, mice were injected with vehicle (T + V) or EXT418 daily (T + 418 Daily, 0.25 mg/kg/day) or every other day (T + 418 EOD, 0.5 mg/kg/EOD) for up to 14 days, whereas HK-treated mice were given vehicle (HK + V). Subsets of T + 418 Daily or EOD-treated mice were pair-fed to the T + V group. Body composition and grip strength were evaluated before tumour implantation and at the end of the experiment. Molecular markers were probed in muscles upon termination. RESULTS: In tumour-bearing mice, administration of EXT418 daily or EOD partially prevented weight loss (T + V vs. T + 418 Daily, P = 0.030; and vs. T + 418 EOD, P = 0.020). Similar effects were observed in whole body fat and lean body mass. Grip strength in tumour-bearing mice was improved by EXT418 daily (P = 0.010) or EOD (P = 0.008) administration compared with vehicle-treated mice. These effects of EXT418 on weight and grip strength were partially independent of food intake. EXT418 daily administration also improved type IIA (P = 0.015), IIB (P = 0.037) and IIX (P = 0.050) fibre cross-sectional area (CSA) in tibialis anterior (TA) and EXT418 EOD improved CSA of IIB fibres in red gastrocnemius (GAS; P = 0.005). In skeletal muscles, tumour-induced increases in atrogenes Fbxo32 and Trim63 were ameliorated by EXT418 treatments (TA and GAS/plantaris, PL), which were independent of food intake. EXT418 administration decreased expression of the mitophagy marker Bnip3 (GAS/PL; P ≤ 0.010). Similar effects of EXT418 EOD were observed in p62 (GAS/PL; P = 0.039). In addition, EXT418 treatments ameliorated the tumour-induced elevation in muscle Il6 transcript levels (TA and GAS/PL), independently of food intake. Il-6 transcript levels in adipose tissue and circulating IL-10 were elevated in response to the tumour but these increases were not significant with EXT418 administration. Tumour mass was not altered by EXT418. CONCLUSIONS: EXT418 mitigates LLC-induced cachexia by attenuating skeletal muscle inflammation, proteolysis, and mitophagy, without affecting tumour mass and partially independent of food intake.

Cisplatin and Albumin-Based Gold-Cisplatin Nanoparticles Enhance Ablative Radiation Therapy-Induced Antitumor Immunity in Local and Distant Tumor Microenvironment.

PURPOSE: Ablative radiation therapy (RT) is an important strategy to eliminate primary tumor and can potentially induce the abscopal effect. Human serum albumin nanoparticle (NP) was used for controlled release of cisplatin to decrease cisplatin's systemic toxicity, and gold (Au) was added to increase RT-induced immunogenic cell death and potentiate the abscopal antitumor immunity. METHODS AND MATERIALS: The designed albumin-based cisplatin-conjugated AuNPs were administered concurrently with ablative RT. C57BL/6 mice implanted with syngeneic murine Lewis lung carcinoma or murine MB49 tumor models were treated with ablative RT (12 Gy per fraction for 2 fractions, total 24 Gy), cisplatin, or Au-cisplatin NPs. RESULTS: Combining ablative RT with cisplatin or Au-cisplatin NPs both destroyed the primary tumor effectively and elicited immunogenic cell death accompanied by release of danger-associated molecular patterns. This enhanced recruitment of effector tumor-infiltrating immune cells, including natural killer T cells and CD8+ T cells, and elicited an increased percentage of professional antigen-presenting CD11c+ dendritic cells. Transient weight loss, accompanying hepatotoxicity, nephrotoxicity, and hematopoietic suppression, was observed as a systemic adverse event in the cisplatin but not the Au-cisplatin NPs group. Cisplatin and Au-cisplatin NPs both showed equivalent ability to reduce metastatic potential when combined with ablative RT, confirmed by suppressed unirradiated flank tumor growth and decreased metastatic lung tumor burden, which translated to improved survival. Mobilization and abundance of effector tumor-infiltrating immune cells including CD8+ T cells and dendritic cells were observed in the distant lung tumor microenvironment after ablative RT with cisplatin or Au-cisplatin NPs, demonstrating increased antitumor immunotherapeutic activity as an abscopal effect. CONCLUSIONS: Compared with cisplatin, the albumin-based Au-cisplatin NPs exhibited equivalent but no superior antitumor immunotherapeutic activity while reducing systemic adverse events and can be safely administered concurrently with ablative RT. Alternative NP formulations may be designed to further improve anticancer outcomes.

Angiogenic inhibitor pre-administration improves the therapeutic effects of immunotherapy.

In lung cancer, immune checkpoint inhibitors (ICIs) are often inadequate for tumor growth inhibition. Angiogenic inhibitors (AIs) are required to normalize tumor vasculature for improved immune cell infiltration. However, in clinical practice, ICIs and cytotoxic antineoplastic agents are simultaneously administered with an AI when tumor vessels are abnormal. Therefore, we examined the effects of pre-administering an AI for lung cancer immunotherapy in a mouse lung cancer model. Using DC101, an anti-vascular endothelial growth factor receptor 2 (VEGFR2) monoclonal antibody, a murine subcutaneous Lewis lung cancer (LLC) model was used to determine the timing of vascular normalization. Microvessel density (MVD), pericyte coverage, tissue hypoxia, and CD8-positive cell infiltration were analyzed. The effects of an ICI and paclitaxel after DC101 pre-administration were investigated. On Day 3, increased pericyte coverage and alleviated tumor hypoxia represented the highest vascular normalization. CD8+ T-cell infiltration was also highest on Day 3. When combined with an ICI, DC101 pre-administration significantly reduced PD-L1 expression. When combined with an ICI and paclitaxel, only DC101 pre-administration significantly inhibited tumor growth, but simultaneous administration did not. AI pre-administration, and not simultaneous administration, may increase the therapeutic effects of ICIs due to improved immune cell infiltration.

Dihydroartemisinin elicits immunogenic death through ferroptosis-triggered ER stress and DNA damage for lung cancer immunotherapy.

BACKGROUND: The immunosuppressive microenvironment of lung cancer serves as an important endogenous contributor to treatment failure. The present study aimed to demonstrate the promotive effect of DHA on immunogenic cell death (ICD) in lung cancer as well as the mechanism. METHODS: The lewis lung cancer cells (LLC), A549 cells and LLC-bearing mice were applied as the lung cancer model. The apoptosis, ferroptosis assay, western blotting, immunofluorescent staining, qPCR, comet assay, flow cytometry, confocal microscopy, transmission electron microscopy and immunohistochemistry were conducted to analyze the functions and the underlying mechanism. RESULTS: An increased apoptosis rate and immunogenicity were detected in DHA-treated LLC and tumor grafts. Further findings showed DHA caused lipid peroxide (LPO) accumulation, thereby initiating ferroptosis. DHA stimulated cellular endoplasmic reticulum (ER) stress and DNA damage simultaneously. However, the ER stress and DNA damage induced by DHA could be abolished by ferroptosis inhibitors, whose immunogenicity enhancement was synchronously attenuated. In contrast, the addition of exogenous iron ions further improved the immunogenicity induced by DHA accompanied by enhanced ER stress and DNA damage. The enhanced immunogenicity could be abated by ER stress and DNA damage inhibitors as well. Finally, DHA activated immunocytes and exhibited excellent anti-cancer efficacy in LLC-bearing mice. CONCLUSIONS: In summary, the current study demonstrates that DHA triggers ferroptosis, facilitating the ICD of lung cancer thereupon. This work reveals for the first time the effect and underlying mechanism by which DHA induces ICD of cancer cells, providing novel insights into the regulation of the immune microenvironment for cancer immunotherapy by Chinese medicine phytopharmaceuticals.

Influence of Tyrosyl-DNA Phosphodiesterase 1 Inhibitor on the Proapoptotic and Genotoxic Effects of Anticancer Agent Topotecan.

To date, various strategies have been proposed to increase the efficiency of cancer therapy. It is known that the action of DNA repair system can determine the resistance of cancer cells to DNA-damaging chemotherapy and radiotherapy, and one of these ways to increase therapeutic efficiency is the search for inhibitors of enzymes of the DNA repair system. Inhibition of the DNA repair enzyme tyrosyl-DNA phosphodiesterase1 (Tdp1) leads to an increase in the effectiveness of the topoisomerase 1 (Top1) inhibitor, the anticancer drug topotecan. Covalent complexes Top1-DNA, which are normally short-lived and are not a threat to the cell, are stabilized under the influence of topotecan and lead to cell death. Tdp1 eliminates such stabilized complexes and thus weaken the effect of topotecan therapy. We have previously shown that the use of the usnic acid hydrazonothiazole derivative OL9-119 in combination with topotecan increased the antitumor and antimetastatic efficacy of the latter in a mouse model of Lewis lung carcinoma. In this work, it was shown that the combined use of topotecan and Tdp1 inhibitor, the hydrazonothiazole derivative of usnic acid OL9-119, leads to an increase in the DNA-damaging effect of topotecan which is used in the clinic for the treatment of cancer. The study of the proapoptotic effect of the compound OL9-119 showed that the compound itself does not induce apoptosis, but increases the proapoptotic effect of topotecan. The results of the study could be used to improve the effectiveness of anticancer therapy and/or to reduce the therapeutic dose of topotecan and, therefore, the severity of side effects.

Low- dose Apatinib promotes vascular normalization and hypoxia reduction and sensitizes radiotherapy in lung cancer.

BACKGROUND AND PURPOSE: Abnormal vascular network of tumor can create a hypoxic microenvironment, and reduce radiotherapy sensitivity. Normalization of tumor vasculature can be a new therapeutic strategy for sensitizing radiotherapy. This study aimed to explore the effect of apatinib on vascular normalization, as well as the syngeneic effect with radiotherapy on lung cancer. MATERIALS AND METHODS: Lewis lung carcinoma (LLC) xenograft-bearing female C57BL/6 mice were treated with different doses of apatinib (30, 60, and 120 mg/kg per day) and/or radiation therapy (8 Gy/1F) and then sacrificed to harvest tumor tissue for immunohistochemical test. Further 18 F-FMISO micro- PET in vivo explored the degree of hypoxia. RESULTS: Immunohistochemistry of CD31 and alpha-smooth muscle actin (α-SMA) proved that low-dose apatinib can normalize vasculature in tumor, especially on Day 10. Tissue staining of hypoxyprobe-1 and 18 F-FMISO micro- PET in vivo showed that 60 mg/kg/day of apatinib significantly alleviates hypoxia. Moreover, this study further proved that low-dose apatinib (60 mg/kg/day) can enhance the radio-response of LLC xenograft mice. CONCLUSION: Our data suggested that low- dose apatinib can successfully induce a vascular normalization window and function as a radio- sensitizer in the lung cancer xenografts model.

EFFECT OF B. SUBTILIS ІМV B-7724 LECTIN ON THE ACTIVITY OF EFFECTORS OF CELLULAR ANTITUMOR IMMUNITY OF MICE WITH LEWIS LUNG CARCINOMA.

AIM: To evaluate the effect of B. subtilis IMV B-7724 lectin on the functional activity of macrophages (Mph), natural killer (NK) cells and cytotoxic lymphocytes (CTL) of mice bearing Lewis lung carcinoma (LLC). MATERIALS AND METHODS: The studies were performed on C57Bl/6J mice; LLC was used as an experimental transplantable tumor. The lectin from B. subtilis IMV B-7724 was administered to LLC-bearing mice subcutaneously at a dose of 1 mg/kg of body weight for 10 days. The immunological testing was performed on days 14, 21, and 28 after tumor grafting. The cytotoxic activity of Mph, NK, and CTL was estimated in MTT-assay; the content of the stable metabolites of nitric oxide (NO) was measured by a standard Griess reaction; the arginase activity (Arg) was determined based on the measurement of urea. RESULTS: The administration of the B. subtilis IMV B-7724 lectin to LLC-bearing mice exerted its antitumor and antimetastatic effects partially via a significant (p < 0.05) increase of Mph and NK activities after the completion of the treatment. In the group of animals injected with lectin, the NO/Arg ratio increased significantly, indicating the prevalence of Mph with proinflammatory and antitumor properties. The cytotoxic activity of Mph exceeded the indices of untreated mice and intact control by 1.8 times and 5.3 times respectively; of NK - by 2.8 and 1.3 times respectively. The effect of treatment on the CTL activity was less pronounced. CONCLUSION: Antitumor and antimetastatic activity of the lectin from B. subtilis IMV B-7724 ensured the preservation of the cytotoxic activity of the main effectors of antitumor immunity (Mph, NK, and CTL) throughout LLC growth.

Immunomodulation of HDAC Inhibitor Entinostat Potentiates the Anticancer Effects of Radiation and PD-1 Blockade in the Murine Lewis Lung Carcinoma Model.

Although the combination of radiotherapy and immunotherapy has proven to be effective in lung cancer treatment, it may not be sufficient to fully activate the antitumor immune response. Here, we investigated whether entinostat, a histone deacetylase inhibitor, could improve the efficacy of radiotherapy and anti-PD-1 in a murine syngeneic LL/2 tumor model. A total of 12 Gy of X-rays administered in two fractions significantly delayed tumor growth in mice, which was further enhanced by oral entinostat administration. Flow cytometry-aided immune cell profiling revealed that entinostat increased radiation-induced infiltration of myeloid-derived suppressor cells and CD8+ T cells with decreased regulatory T-cells (Tregs). Transcriptomics-based immune phenotype prediction showed that entinostat potentiated radiation-activated pathways, such as JAK/STAT3/interferon-gamma (IFN-γ) and PD-1/PD-L1 signaling. Entinostat augmented the antitumor efficacy of radiation and anti-PD-1, which may be related to an increase in IFN-γ-producing CD8+ T-cells with a decrease in Treg cells. Comparative transcriptomic profiling predicted that entinostat increased the number of dendritic cells, B cells, and T cells in tumors treated with radiation and anti-PD-1 by inducing MHC-II genes. In conclusion, our findings provided insights into how entinostat improves the efficacy of ionizing radiation plus anti-PD-1 therapy and offered clues for developing new strategies for clinical trials.

Targeting EGFR in Combination with Nutritional Supplements on Antitumor Efficacy in a Lung Cancer Mouse Model.

Selenium (Se) and fish oil (FO) exert anti-epidermal growth factor receptor (EGFR) action on tumors. This study aimed to compare the anti-cancer efficacy of EGFR inhibitors (gefitinib and erlotinib) alone and in combination with nutritional supplements of Se/FO in treating lung cancer. Lewis LLC1 tumor-bearing mice were treated with a vehicle or Se/FO, gefitinib or gefitinib plus Se/FO, and erlotinib or erlotinib plus Se/FO. The tumors were assessed for mRNA and protein expressions of relevant signaling molecules. Untreated tumor-bearing mice had the lowest body weight and highest tumor weight and volume of all the mice. Mice receiving the combination treatment with Se/FO and gefitinib or erlotinib had a lower tumor volume and weight and fewer metastases than did those treated with gefitinib or erlotinib alone. The combination treatment exhibited greater alterations in receptor signaling molecules (lower EGFR/TGF-ß/TßR/AXL/Wnt3a/Wnt5a/FZD7/ß-catenin; higher GSK-3ß) and immune checkpoint molecules (lower PD-1/PD-L1/CD80/CTLA-4/IL-6; higher NKp46/CD16/CD28/IL-2). These mouse tumors also had lower angiogenesis, cancer stemness, epithelial to mesenchymal transitions, metastases, and proliferation of Ki-67, as well as higher cell cycle arrest and apoptosis. These preliminary results showed the Se/FO treatment enhanced the therapeutic efficacies of gefitinib and erlotinib via modulating multiple signaling pathways in an LLC1-bearing mouse model.

Modified Bu-Fei decoction inhibits lung metastasis via suppressing angiopoietin-like 4.

BACKGROUND: Modified Bu-Fei decoction (MBFD), a formula of traditional Chinese medicine, is used for treating lung cancer in clinic. The actions and mechanisms of MBFD on modulating lung microenvironment is not clear. PURPOSE: Lung microenvironment is rich in vascular endothelial cells (ECs). This study is aimed to examine the actions of MBFD on tumor biology, and to uncover the underlying mechanisms by focusing on pulmonary ECs. METHODS: The Lewis lung carcinoma (LLC) xenograft model and the metastatic cancer model were used to determine the efficacy of MBFD on inhibiting tumor growth and metastasis. Flow cytometry and trans-well analysis were used to determine the role of ECs in anti-metastatic actions of MBFD. The in silico analysis and function assays were used to identify the mechanisms of MBFD in retarding lung metastasis. Plasma from lung cancer patients were used to verify the effects of MBFD on angiogenin-like protein 4 (ANGPTL4) in clinical conditions. RESULTS: MBFD significantly suppressed spontaneous lung metastasis of LLC tumors, but not tumor growth, at clinically relevant concentrations. The anti-metastatic effects of MBFD were verified in metastatic cancer models created by intravenous injection of LLC or 4T1 cells. MBFD inhibited lung infiltration of circulating tumor cells, without reducing tumor cell proliferations in lung. In vitro, MBFD dose-dependently inhibited trans-endothelial migrations of tumor cells. RNA-seq assay and verification experiments confirmed that MBFD potently depressed endothelial ANGPTL4 which is able to broke endothelial barrier and protect tumor cells from anoikis. Database analysis revealed that high ANGPTL4 levels is negatively correlated with overall survival of cancer patients. Importantly, MBFD therapy reduced plasma levels of ANGPTL4 in lung cancer patients. Finally, MBFD was revealed to inhibit ANGPTL4 expressions in a hypoxia inducible factor-1α (HIF-1α)-dependent manner, based on results from specific signaling inhibitors and network pharmacology analysis. CONCLUSION: MBFD, at clinically relevant concentrations, inhibits cancer lung metastasis via suppressing endothelial ANGPTL4. These results revealed novel effects and mechanisms of MBFD in treating cancer, and have a significant clinical implication of MBFD therapy in combating metastasis.

АNTIMETASTATIC EFFECT OF B. SUBTILIS IMV B-7724 LECTIN OBSERVED IN LEWIS LUNG CARCINOMA MODEL.

AIM: To study the antitumor and antimetastatic effects of B. subtilis IMV B-7724 lectin used in neoadjuvant and adjuvant settings in vivo. MATERIALS AND METHODS: Studies were performed on C57Bl/6J mice; Lewis lung carcinoma (LLC) was used as an experimental tumor. В. subtilis ІМV В-7724 lectin was administered to tumor-bearing mice or to mice which underwent surgical resection of the primary tumor. The lectin was injected subcutaneously, 10 times, at a single dose of 5 or 1 mg/kg of body weight. The standard indicators of tumor growth and metastasis were evaluated. RESULTS: Independently of the application settings, the lectin at a dose of 1 mg/kg of b.w. caused more pronounced effect than at a dose of 5 mg/kg of b.w. The administration of B. subtilis IMV B-7724 lectin to the mice with LLC in neoadjuvant setting did not cause notable antitumor effect but led to a significant decrease in the number and volume of lung metastases. The lectin administration in adjuvant setting significantly inhibited metastasis: the metastasis inhibition index reached 63.0% and 100% in the mice treated with the lectin at a dose of 5 mg/kg and 1 mg/kg respectively. The mean survival time of the treated animals significantly increased. CONCLUSION: A pronounced antimetastatic effect of B. subtilis IMV B-7724 lectin administered in an adjuvant setting was demonstrated.

Erythropoiesis-Stimulating Properties of Anthocyanin-Containing Complex from Sorbus aucuparia L. in Cytostatic Anemic Syndrome in Mice with Lewis Lung Carcinoma.

The results of studying the effect of the anthocyanin-containing complex from Sorbus aucuparia L. on the main indicators of erythropoiesis in the blood and bone marrow of mice with Lewis lung carcinoma against the background of doxorubicin administration were presented. It was shown that administration of the anthocyanin-containing complex from S. aucuparia L. to animals with the tumor prevented the development of anemic syndrome by promoting regeneration of the erythropoiesis after its depletion caused by single administration of a cytostatic agent.