Mouse Lewis Lung Carcinoma Research Articles

Trichosanthin elicits antitumor activity via MICU3 mediated mitochondria calcium influx

IntroductionTrichosanthin (TK) is a glycoprotein extracted from the Chinese medicinal herb Trichosanthes kirilowi, which has anti-virus and anti-tumor activity. However, the target and detailed mechanism of TK remains elusive. ObjectivesWe aimed to identify novel antitumor targets of TK in lung adenocarcinoma and study its anti-tumor mechanism. MethodsWe utilized a Lewis lung carcinoma mouse model to evaluate the inhibition of TK on tumor growth. CCK8 assay was utilized to calculate IC50 of trichosanthin on A549 and H1299. In-vitro cellular assays and in-vivo xenograft mice studies were used to investigate MICU3 overexpression and TK treatment on tumor growth. Fluo-4 dye and JC-1 staining was used to measure the mitochondrial calcium levels and membrane potential. H&E and immunohistochemistry staining were applied the asses the effect of TK on tumor and microenvironment. RNA sequencing was applied to analyze transcriptome changes in TK-treated and MICU3-overexpressed tumor cells. The influence of trichosanthin on DNMT3B expression and MICU3 methylation were detected by qPCR and Western blotting. Transcriptional activity of the MICU3 gene was measured by ChIP-PCR and luciferase assays. ResultsTrichosanthin ihibited the tumor growth in vivo, resulting cancer cell growth inhibition and cell death, with almost no effect on normal cells. IC50 of trichosanthin in A549 and H1299 cells were 62.8 μg/ml and 39.7 μg/ml, respectively. Mitochondrial Calcium Uptake Family complex MICU3 was shown to associated with favorable prognosis and was upregulated upon trichosanthin treatment, along with reduces tumor cell growth and migration, and increased cell death both in vitro and in vivo. Increased mitochondrial calcium level was observed in MICU3 overexpression cells. Pathway analysis of RNA-seq data revealed that cytokine and receptor pathways were enriched in MICU3-overexpressing cells. Trichosanthin decreased DNMT3B expression and altered MICU3 methylation while increased FOSL2 expression and reduced methylation that correlated with increased transcription of the MICU3 gene. ConclusionTrichosanthin elicits antitumor activity in lung adenocarcinoma via repressing DNMT3B and increasing FOSL2, which in turn induces MICU3-mediated mitochondrial calcium influx and tumor cell death.

Astragaloside IV augments anti-PD-1 therapy to suppress tumor growth in lung cancer by remodeling the tumor microenvironment.

Programmed cell death protein-1 (PD-1) inhibitors are increasingly utilized in the treatment of lung cancer (LC). Combination therapy has recently gained popularity in treating LC. This study aimed to assess the efficacy of combining Astragaloside IV (AS-IV) and anti-PD-1 in LC. C57BL/6J mice were subcutaneously injected with Lewis lung carcinoma (LLC) cells. After 3 weeks, the animals were sacrificed, and the tumors were harvested for analysis. Ki-67 immuno-labeling and TUNEL assay were used for evaluating cell proliferation and apoptosis in tumor tissues. In addition, anti-cleaved caspase 3 was used for immunolabelling of apoptotic cells. Immune cell infiltration (macrophages and T cells) and gene expression in tumor tissues were also investigated by using immunofluorescence staining. Compared to treatment with anti-PD-1 or AS-IV, the combination of AS-IV and anti-PD-1 notably reduced tumor volume and weight of LLC-bearing mice. Additionally, the combination treatment strongly induced the apoptosis and suppressed the proliferation in tumor tissues through inactivating PI3K/Akt and ERK signaling pathways, compared to single treatment group. Moreover, the combination treatment elevated levels of the M1 macrophage marker mCD86, reduced levels of the M2 macrophage marker mCD206, as well as upregulated levels of the T cell activation marker mCD69 in tumor tissues. Collectively, the combination treatment effectively inhibited tumor growth in LLC mice through promoting M1 macrophage polarization and T cell activation. These findings showed that combining AS-IV with anti-PD-1 therapy could be a promising therapeutic approach for LC.

Alleviating tumor hypoxia and immunosuppression via sononeoperfusion: A new Ally for potentiating anti-PD-L1 blockade of solid tumor

The hypoxic and immunosuppressive tumor microenvironment (TME) remains a major obstacle to impede cancer immunotherapy. Here, we found that sononeoperfusion—a new effect of tumor perfusion enhancement induced by low mechanical index ultrasound stimulated microbubble cavitation (USMC)—ameliorated tumor tissue oxygenation and induced tumor vascular normalization (TVN). This TVN might be associated with the down-regulation of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) within tumors. Moreover, the sononeoperfusion effect reduced the accumulation of immunosuppressive cells, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and M2-like tumor-associated macrophages (M2-TAMs), and decreased the production of immune inhibitory factors like transforming growth factor-β1 (TGF-β1), interleukin 10 (IL-10), chemoattractant chemokines CC-chemokine ligand 22 (CCL22), CCL28, adenosine and lactate within tumors. Notably, flow cytometry analysis revealed that sononeoperfusion not only increased the percentage of tumor infiltrating-CD8+ T cells, but also promoted the generation of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) by these cells. Furthermore, the improved immune TME by sononeoperfusion effect sensitized anti-PD-L1 treatment both in MC38 colon cancer and Lewis lung carcinoma mice, resulting in tumor regression and prolonged survival. Mechanically, the enhanced efficacy of combination therapy was mainly based on promoting the infiltration and function of CD8+ T cells within tumors. Together, sononeoperfusion could ameliorate hypoxia and immunosuppression in the TME, thereby potentiating anti-PD-L1 therapy for solid tumors. This novel method of USMC generating sononeoperfusion effect may provide a new therapeutic modality for facilitating cancer immunotherapy.

P3.02F.06 Multi-Omics Reveals Effects of Gut Microbiome on Tumor Immune and Metabolism in Mouse Lewis Lung Carcinoma Model

P3.02F.06 Multi-Omics Reveals Effects of Gut Microbiome on Tumor Immune and Metabolism in Mouse Lewis Lung Carcinoma Model

The LEAP2 Response to Cancer-Related Anorexia-Cachexia Syndrome in Male Mice and Patients.

The hormone ghrelin serves a protective role in cancer-related anorexia-cachexia syndrome (CACS)-a condition in which plasma levels of ghrelin rise, its administration lessens CACS severity, and experimentally reduced signaling by its receptor (GHSR) worsens fat loss and anorexia and accelerates death. Yet, actions for the related hormone liver-expressed antimicrobial peptide-2 (LEAP2), which is an endogenous GHSR antagonist, are unexplored in CACS. Here, we found that plasma LEAP2 and LEAP2/ghrelin ratio were lower in Lewis lung carcinoma (LLC) and RM-9 prostate cancer CACS mouse models. Ghrelin deletion exaggerated losses of tumor-free body weight and fat mass, reduced food intake, reduced soleus muscle weight, and/or lowered grip strength in LLC or RM-9 tumor-bearing mice. LEAP2 deletion lessened reductions in tumor-free body weight and fat mass and increased food intake in LLC or RM-9 tumor-bearing mice. In a 55-subject cohort of patients with CACS or weight-stable cancer, the plasma LEAP2/total ghrelin ratio was negatively correlated with 6-month weight change preceding blood collection. These data demonstrate that ghrelin deletion exacerbates CACS in the LLC and RM-9 tumor-bearing mouse models while contrastingly, LEAP2 deletion reduces measures of CACS in these tumor-bearing mouse models. Further, they suggest that lower plasma LEAP2/ghrelin ratio protects against worsened CACS.

From 2D to 3D In Vitro World: Sonodynamically-Induced Prooxidant Proapoptotic Effects of C60-Berberine Nanocomplex on Cancer Cells.

The primary objective of this research targeted the biochemical effects of SDT on human cervix carcinoma (HeLa) and mouse Lewis lung carcinoma (LLC) cells grown in 2D monolayer and 3D spheroid cell culture. HeLa and LLC monolayers and spheroids were treated with a 20 µM C60-Ber for 24 h, followed by irradiation with 1 MHz, 1 W/cm2 US. To evaluate the efficacy of the proposed treatment on cancer cells, assessments of cell viability, caspase 3/7 activity, ATP levels, and ROS levels were conducted. Our results revealed that US irradiation alone had negligible effects on LLC and HeLa cancer cells. However, both monolayers and spheroids irradiated with US in the presence of the C60-Ber exhibited a significant decrease in viability (32% and 37%) and ATP levels (42% and 64%), along with a notable increase in ROS levels (398% and 396%) and caspase 3/7 activity (437% and 246%), for HeLa monolayers and spheroids, respectively. Similar tendencies were observed with LLC cells. In addition, the anticancer effects of C60-Ber surpassed those of C60, Ber, or their mixture (C60 + Ber) in both cell lines. The detected intensified ROS generation and ATP level drop point to mitochondria dysfunction, while increased caspase 3/7 activity points on the apoptotic pathway induction. The combination of 1 W/cm2 US with C60-Ber showcased a promising platform for synergistic sonodynamic chemotherapy for cancer treatment.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol treatment inhibits abnormal tumor growth by regulating neutrophil infiltration in a non-small cell lung carcinoma mouse model

Excessive neutrophil infiltration into the tumor microenvironment (TME) is an important factor that contributes to tumor overgrowth and limited immunotherapy efficacy. Neutrophils activate various receptors involved in tumor progression, while suppressing the infiltration and activity of cytotoxic T cells and creating optimal conditions for tumor growth. Therefore, the appropriate control of neutrophil infiltration is an effective strategy for tumor treatment.In the present study, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) inhibited tumor overgrowth by suppressing excessive neutrophil infiltration, resulting in >74.97 % reduction in tumor size in a Lewis lung carcinoma (LLC-1) mouse model. All subjects in the positive control group died during the 90-day survival period, whereas only four subjects in the PLAG treatment group survived. PLAG had a significantly higher tumor growth inhibitory effect and survival rate than other neutrophil infiltration-targeting inhibitors (e.g., Navarixin, lymphocyte antigen 6 complex locus G6D antibody [aLy6G]). The ability of PLAG to regulate neutrophil infiltration and inhibit tumor growth depends on thioredoxin-interacting protein (TXNIP). In tumors lacking TXNIP expression, PLAG failed to control neutrophil infiltration and infiltration-related factor release, and the inhibitory effect of PLAG on tumor growth was reduced. PLAG-mediated inhibition of neutrophil infiltration enhances the efficacy of immune checkpoint inhibitors (ICIs), increasing the antitumor efficacy and survival rate by 30 %.In conclusion, PLAG could be a novel alternative to anti-tumor drugs that effectively targets excessive neutrophil infiltration into cancer tissues.

Antitumor Study of Neoantigen-reactive T Cells Co-expressing IL-7 and CCL19 in Mouse Lung Cancer

Neoantigen reactive T cell (NRT) has the ability to inhibit the growth of tumors expressing specific neoantigens. However, due to the difficult immune infiltration and the inhibition of tumor microenvironment, the therapeutic effect of NRT in solid tumors is limited. In this study, we designed NRT cells (7×19 NRT) that can express both interleukin-7 (IL-7) and chemokine C-C motif ligand 19 (CCL19) in mouse lung cancer cells, and evaluated the difference in anti-tumor effect between 7×19 NRT cells and conventional NRT cells. We performed next-generation sequencing and neoantigen prediction for mouse Lewis lung carcinoma (LLC), prepared RNA vaccine, cultured NRT cells, constructed retroviral vectors encoding IL-7 and CCL19, transduced NRT cells and IL-7 and CCL19 were successfully expressed, and 7×19 NRT was successfully obtained. The anti-tumor effect was evaluated in vivo and in vitro in mice. The 7×19 NRT cells significantly enhanced the proliferation and invasion ability of T cells by secreting IL-7 and CCL19, achieved significant tumor inhibition in the mouse lung cancer and extended the survival period of mice. The T cell infiltration into tumor tissue and the necrosis of tumor tissue increased significantly after 7×19 NRT treatment. In addition, both 7×19 NRT treatment and conventional NRT treatment were safe. The anti-solid tumor ability of NRT cells is significantly enhanced by the arming of IL-7 and CCL19, which is a safe and effective genetic modification of NRT.

Decreased skeletal muscle intramyocellular lipid droplet-mitochondrial contact contributes to myosteatosis in cancer cachexia.

Cancer cachexia, the unintentional loss of lean mass, contributes to functional dependency, poor treatment outcomes, and decreased survival. Although its pathogenicity is multifactorial, metabolic dysfunction remains a hallmark of cachexia. However, significant knowledge gaps exist in understanding the role of skeletal muscle lipid metabolism and dynamics in this condition. We examined skeletal muscle metabolic dysfunction, intramyocellular lipid droplet (LD) content, LD morphology and subcellular distribution, and LD-mitochondrial interactions using the Lewis lung carcinoma (LLC) murine model of cachexia. C57/BL6 male mice (n = 20) were implanted with LLC cells (106) in the right flank or underwent PBS sham injections. Skeletal muscle was excised for transmission electron microscopy (TEM; soleus), oil red O/lipid staining [tibialis anterior (TA)], and protein (gastrocnemius). LLC mice had a greater number (232%; P = 0.006) and size (130%; P = 0.023) of intramyocellular LDs further supported by increased oil-red O positive (87%; P = 0.0109) and "very high" oil-red O positive (178%; P = 0.0002) fibers compared with controls and this was inversely correlated with fiber size (R2 = 0.5294; P < 0.0001). Morphological analyses of LDs show increased elongation and complexity [aspect ratio: intermyofibrillar (IMF) = 9%, P = 0.046) with decreases in circularity [circularity: subsarcolemmal (SS) = 6%, P = 0.042] or roundness (roundness: whole = 10%, P = 0.033; IMF = 8%, P = 0.038) as well as decreased LD-mitochondria touch (-15%; P = 0.006), contact length (-38%; P = 0.036), and relative contact (86%; P = 0.004). Furthermore, dysregulation in lipid metabolism (adiponectin, CPT1b) and LD-associated proteins, perilipin-2 and perilipin-5, in cachectic muscle (P < 0.05) were observed. Collectively, we provide evidence that skeletal muscle myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in a preclinical model of cancer cachexia.NEW & NOTEWORTHY We sought to advance our understanding of skeletal muscle lipid metabolism and dynamics in cancer cachexia. Cachexia increased the number and size of intramyocellular lipid droplets (LDs). Furthermore, decreases in LD-mitochondrial touch, contact length, and relative contact along with increased LD shape complexity with decreases in circularity and roundness. Dysregulation in lipid metabolism and LD-associated proteins was also documented. Collectively, we show that myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in cancer cachexia.

Structural Elucidation and Anti-Tumor Activity of a Polysaccharide (CP2-S) from Cordyceps militaris Fruit Bodies.

A polysaccharide (CP2-S), consisting of glucose with a weight average molecular weight of 5.9 × 106, was purified from the fruit bodies of Cordyceps militaris. In this work, the corresponding structure and anti-tumor activity in vivo were investigated. Methylation and NMR analysis revealed that CP2-S was composed of a →4)-α-D-Glcp-(1→ backbone with partial substitution occurring at O-6 by T-linked α-D-Glcp in every ten residues, which has not been reported in previous reports. In vivo anti-tumor experiments showed that CP2-S could inhibit the growth of Lewis lung carcinoma in mice. Tumor inhibition rates were 17.8%, 24.5%, and 29.5% at dosages of 12.5, 50, and 100 mg/kg/d, respectively. Compared with the cisplatin group, mice treated with CP2-S exhibited a significant increase in spleen index (increased 22.7-42.4%) and thymus index (increased 47.7-36.8%). Additionally, serum levels of IgM and IgG in tumor-bearing mice increased by approximately 6.11~10.75-folds and 1.31~1.38-folds, respectively. These findings prove that CP2-S significantly inhibited the growth of Lewis lung carcinoma through immune-enhancing activity in mice.

Targeting metabolic pathways alleviates bortezomib-induced neuropathic pain without compromising anticancer efficacy in a sex-specific manner.

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect of cancer treatment that significantly impacts patients' quality of life. This study investigated the effects of targeting metabolic pathways on bortezomib-induced neuropathic pain and tumor growth using a Lewis lung carcinoma (LLC) mouse model, while exploring potential sex differences. Male and female C57BL/6J mice were implanted with LLC cells and treated with bortezomib alone or in combination with metformin, dichloroacetate (DCA), or oxamate. Tactile allodynia was assessed using von Frey filaments. Tumor volume and weight were measured to evaluate tumor growth. Metformin, DCA, and oxamate effectively attenuated bortezomib-induced neuropathic pain without compromising the anticancer efficacy of bortezomib in both male and female mice. The LLC model exhibited a paraneoplastic neuropathy-like phenotype. Significant sex differences were observed, with male mice exhibiting larger tumors compared to females. Oxamate was more effective in alleviating allodynia in males, while metformin and DCA showed greater efficacy in reducing tumor growth in females. Targeting metabolic pathways can alleviate CIPN without interfering with bortezomib's anticancer effects. The LLC model may serve as a tool for studying paraneoplastic neuropathy. Sex differences in tumor growth and response to metabolic interventions highlight the importance of considering sex as a biological variable in preclinical and clinical studies investigating cancer biology, CIPN, and potential therapeutic interventions.

Tumor promoting effect of PDLIM2 downregulation involves mitochondrial ROS, oncometabolite accumulations and HIF-1α activation

BackgroundCancer is characterized by dysregulated cellular metabolism. Thus, understanding the mechanisms underlying these metabolic alterations is important for developing targeted therapies. In this study, we investigated the pro-tumoral effect of PDZ and LIM domain 2 (PDLIM2) downregulation in lung cancer growth and its association with the accumulation of mitochondrial ROS, oncometabolites and the activation of hypoxia-inducible factor-1 (HIF-1) α in the process.MethodsDatabases and human cancer tissue samples were analyzed to investigate the roles of PDLIM2 and HIF-1α in cancer growth. DNA microarray and gene ontology enrichment analyses were performed to determine the cellular functions of PDLIM2. Seahorse assay, flow cytometric analysis, and confocal microscopic analysis were employed to study mitochondrial functions. Oncometabolites were analyzed using liquid chromatography–mass spectrometry (LC–MS). A Lewis lung carcinoma (LLC) mouse model was established to assess the in vivo function of PDLIM2 and HIF-1α.ResultsThe expression of PDLIM2 was downregulated in lung cancer, and this downregulation correlated with poor prognosis in patients. PDLIM2 highly regulated genes associated with mitochondrial functions. Mechanistically, PDLIM2 downregulation resulted in NF-κB activation, impaired expression of tricarboxylic acid (TCA) cycle genes particularly the succinate dehydrogenase (SDH) genes, and mitochondrial dysfunction. This disturbance contributed to the accumulation of succinate and other oncometabolites, as well as the buildup of mitochondrial reactive oxygen species (mtROS), leading to the activation of hypoxia-inducible factor 1α (HIF-1α). Furthermore, the expression of HIF-1α was increased in all stages of lung cancer. The expression of PDLIM2 and HIF-1α was reversely correlated in lung cancer patients. In the animal study, the orally administered HIF-1α inhibitor, PX-478, significantly reduces PDLIM2 knockdown-promoted tumor growth.ConclusionThese findings shed light on the complex action of PDLIM2 on mitochondria and HIF-1α activities in lung cancer, emphasizing the role of HIF-1α in the tumor-promoting effect of PDLIM2 downregulation. Additionally, they provide new insights into a strategy for precise targeted treatment by suggesting that HIF-1α inhibitors may serve as therapy for lung cancer patients with PDLIM2 downregulation.

Inhibition of ALDOA‐ and ENO1‐Mediated Glycolysis of Oridonin as a Novel Antitumor Strategy of Non‐Small‐Cell Lung Cancer

AbstractNon‐small‐cell lung cancer (NSCLC) is the most common type of lung cancer. Oridonin (ORI), the main pharmacodynamic component of the Chinese herb Herba Rabdosiae with anticancer activity is reported to inhibit various types of cancer including NSCLC. However, its direct target proteins acting on NSCLC still require further investigation. Here, the direct protein profile is analyzed and the key targets of ORI are identified by combining activity‐based protein profiling (ABPP) and the cellular thermal shift assay (CETSA). As a result, ORI can significantly promote Lewis Lung Carcinoma (LLC) cell apoptosis, decrease mitochondrial membrane potential, and increase reactive oxygen species accumulation. In addition, both ABPP and CETSA indicate that ORI directly binds to key proteins of the glycolysis pathway, including Aldolase A (ALDOA), Enolase 1 (ENO1), pyruvate kinase M2, and lactate dehydrogenase. Based on the LLC mouse model, ORI markedly inhibits the growth of lung tumors, increases the serum levels of antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase, and reduces the production of malondialdehyde. To sum up, ORI can be regarded as a novel antitumor strategy of NSCLC by inhibiting ALDOA‐ and ENO1‐mediated glycolysis.

Muscle p62 stimulates the expression of antioxidant proteins alleviating cancer cachexia

Cancer cachexia is characterized by a significant reduction in body weight predominantly resulting from severe loss of skeletal muscle. Oxidative stress plays an important role in skeletal muscle atrophy during cancer cachexia, and more glycolytic muscles are preferentially affected. Sequestosome1/SQSTM1 (i.e., p62), particularly when phosphorylated at Ser 349 (Ser 351 in mice), competitively binds to the Kelch-like ECH-associated protein 1 (Keap1) activating Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 then stimulates the transcription of antioxidant/electrophile-responsive elements (ARE/EpRE) in target genes. However, a potential role for p62 in the protection of muscle wasting in cachexia remains to be determined. Here, using the well-established cachexia-inducing model of Lewis Lung Carcinoma (LLC) in mice we demonstrate higher expression of antioxidant proteins (i.e., NQO1, HO-1, GSTM1, CuZnSOD, MnSOD, and EcSOD) in the more oxidative and cachexia resistant soleus muscle than in the more glycolytic and cachexia prone extensor digitorum longus muscle. This was accompanied by higher p62 (total and phosphorylated) and nuclear Nrf2 levels in the soleus, which were paralleled by higher expression of proteins known to either phosphorylate or promote p62 phosphorylation (i.e., NBR1, CK1, PKCδ, and TAK1). Muscle-specific p62 gain-of-function (i.e., in p62 mTg mice) activated Nrf2 nuclear translocation and increased the expression of multiple antioxidant proteins (i.e., CuZnSOD, MnSOD, EcSOD, NQO1, and GSTM1) in glycolytic muscles. Interestingly, skeletal muscle Nrf2 haplodeficiency blunted the increases of most of these proteins (i.e., CuZnSOD, EcSOD, and NQO1) suggesting that muscle p62 stimulates antioxidant protein expression also via additional, yet to be determined mechanisms. Of note, p62 gain-of-function mitigated glycolytic muscle wasting in LLC-affected mice. Collectively, our findings identify skeletal muscle p62 as a potential therapeutic target for cancer cachexia. This research was supported by Grant-in-Aid for JSPS Fellows (20J15551), Grant-in-Aid for Early-Career Scientists (22K17733), Suzuken Memorial Foundation, The Nakatomi Foundation, and The Uehara Memorial Foundation (to M.Y.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Expression of the TGF-β Target Gene Mss51 Is Induced in Muscle From Tumor-Bearing Mice and Attenuated by Treatment With the AMPK Activator AICAR

Cachexia is a severe complication of cancer characterized by skeletal muscle atrophy and loss of function and is associated with a poor prognosis. AMPK is a cellular energy sensor that is upregulated in cancer cachexia. AMPK activation is well-established in promoting catabolic cell signaling in skeletal muscle, partly through the induction of muscle-specific ubiquitin ligases MAFBx and MuRF-1 expression. Despite AMPK’s acute catabolic effects, chronic treatment with the AMPK-activating drug 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has been reported to preserve muscle function and metabolism in various muscle-wasting conditions, including cancer cachexia. Elevated TGF-β during cancer cachexia is known to disrupt mitochondrial homeostasis and lead to mitochondrial dysfunction. Mss51 is a skeletal muscle-specific mitochondrial gene that is upregulated through TGF-β signaling. Although the role of Mss51 in skeletal muscle cancer cachexia has not been studied as of yet, its expression is associated with mitochondrial and metabolic impairment. Therefore, we hypothesized that Mss-51 expression would be elevated in cachexic skeletal muscle and attenuated by chronic AMPK activation. To test this, we inoculated mice (n=4) with either Lewis Lung Carcinoma (LLC) cells or PBS. Mice were injected daily with 350 mg/kg AICAR or an equivalent volume of saline beginning seven days after inoculation. Twenty-four days after inoculation, the mice were euthanized, and tissues were harvested and weighed. RNA was isolated from the gastrocnemius-plantaris-soleus complex and analyzed by RT-PCR for Mss51, MuRF-1, and MAFBx expression. Protein expression for hexokinase II (HKII) was measured by western blotting. AICAR treatment did not attenuate muscle atrophy in tumor-bearing mice. Compared to the control, Mss-51 was elevated in LLC mice treated with saline and decreased in LLC mice treated with AICAR, consistent with the known effects of AMPK in blocking TGF-β signaling. Similarly, MuRF-1 and MAFBx tended to be elevated in LLC-saline. Still, this increase was attenuated in LLC-AICAR mice, though we were likely underpowered to detect significance in this measure. In conclusion, based on previous work, the rise in Mss51 in tumor-bearing mice may contribute to mitochondrial and metabolic dysfunction in cancer cachexia. The prevention of this increase by AICAR suggests that AMPK may benefit dysfunctional metabolism in the cachexic muscle through inhibition of Mss51. Funding for these experiments was from a BYU Widtsoe Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Influence of allogeneic mesenchymal stem cells of red bone marrow culture on the development of Lewis lung epidermoid carcinoma in vivo

The relevance of this study is conditioned by the widespread use of stem cells in veterinary medicine, a wide range of studies and ambiguous data on the oncoprotective properties of stem cells of different origins. In this regard, the purpose of this study was to investigate the course of the tumour process in Lewis lung carcinoma and the specific features of the effect of allogeneic mesenchymal stem cells of red bone marrow culture on it. The leading approach to investigating this problem was the method of modelling Lewis lung carcinoma in C57BL6 mice and the use of stem cells. The use of allogeneic mesenchymal stem cells from the bone marrow culture of C57BL6 mice with transplanted epidermoid metastatic carcinoma of the Lewis lung contributed to the activation of the tumour process. Under the influence of allogeneic mesenchymal stem cells of red bone marrow culture from Day 14 to Day 24 of the study, the body weight of mice decreased by 7.0-12.1% (P &lt; 0.05) compared to the control, the diameter of the primary tumour increased by 1.43-1.51 times (P &lt; 0.05), which is conditioned by the activation of primary tumour growth. The number of lymphocytes as producers of vascular growth factor in primary tumour tissue under the influence of allogeneic mesenchymal stem cells of red bone marrow culture significantly increased by 1.47 and 1.52 times on Day 18 of the experiment compared to animals of the control group and placebo (P &lt; 0.05), respectively. This promoted angiogenesis in the primary tumour node and metastasis through the circulatory system. After administration allogeneic mesenchymal cells of red bone marrow culture to mice, a larger volume of lung metastases was recorded, which was 41.52±7.9 mm3 compared to the values in the control and placebo groups, respectively, 17.94±6.59 and 16.43±5.32 mm3 . The morphological picture of the histological sections of the primary tumour of Lewis lung carcinoma confirms all the signs of qualitative and quantitative indicators of its progression. The findings obtained are of both theoretical and practical value for clinical veterinary medicine on the use of allogeneic bone marrow mesenchymal stem cells in tumour processes

The use of methylene blue to control the tumor oxygenation level

BackgroundHypoxia is a characteristic feature of many tumors. It promotes tumor proliferation, metastasis, and invasion and can reduce the effectiveness of many types of cancer treatment. ObjectiveThe aim of this study was to investigate the pharmacokinetics of methylene blue (MB) and its impact on the tumor oxygenation level at mouse Lewis lung carcinoma (LLC) model using spectroscopic methods. ApproachThe pharmacokinetics of MB were studied qualitatively and quantitatively using video fluorescence imaging and fluorescence spectroscopy. The degree of hemoglobin oxygenation in vivo was examined by calculating hemoglobin optical absorption from the measured diffuse reflectance spectra. The distribution of MB fluorescence and the lifetime of NADH were analyzed using laser scanning microscopy and fluorescence lifetime imaging microscopy (FLIM) to assess cellular metabolism. ResultsAfter intravenous administration of MB at 10–20 mg/kg, it quickly transitioned in the tumor to a colorless leucomethylene blue, with maximum accumulation in the tumor occurring after 5–10 min. A concentration of 10 mg/kg resulted in a relative increase of the tumor oxygenation level for small tumors (volume 50–75 mm3) and normal tissue 120 min after the introduction of MB. A shift in tumor metabolism towards oxidative phosphorylation (according to the lifetime of the NADH coenzyme) was measured using FLIM method after intravenous administration of 10 mg/kg of MB. Intravenous administration of MB at 20 mg/kg results in a long-term decrease in oxygenation, which persisted for at least 120 min after the administration and did not return to its initial level. ConclusionsAdministration of MB at 10 mg/kg shown to increase tumor oxygenation level, potentially leading to more effective antitumor therapy. However, at higher doses (20 mg/kg), MB may cause long-term decrease in oxygenation.

18β-glycyrrhetinic acid suppresses Lewis lung cancer growth through protecting immune cells from ferroptosis.

18β-glycyrrhetinic acid (GA), the main metabolite of glycyrrhizic acid extracted from the root of licorice, has been reported to possess anti-cancer and immunomodulatory activity, but the mechanisms are not well understood. Recent studies have shown that ferroptosis of immune cells is involved in tumor-associated immune suppression. The purpose of this study was to investigate whether the enhanced immune response via inhibiting immune cell ferroptosis contributed to the anticancer effect of 18β-GA. Lewis Lung carcinoma mouse model and Murine CD8+T cell culture model were used to examine the changes of immune response and ferroptosis of immune cells. We found that 18β-GA was effective against lung cancer accompanied by enhanced activation of tumor-infiltrating CD8+ T cells in Lewis Lung carcinoma mouse model. Furthermore, we demonstrated that the boosted immune response by GA was attributed to its ability to inhibit arachidonic acid (AA)-mediated CD8+ T ferroptosis via suppressing CD36 expression. The findings of the present study unraveled a novel mechanism underlying the anti-cancer and immunomodulatory activity of 18β-GA and support that 18β-GA holds potential to be used as an immune enhancer for lung cancer prevention or treatment.

Obesity worsens mitochondrial quality control and does not protect against skeletal muscle wasting in murine cancer cachexia.

More than 650 million people are obese (BMI>30) worldwide, which increases their risk for several metabolic diseases and cancer. While cachexia and obesity are at opposite ends of the weight spectrum, leading many to suggest a protective effect of obesity against cachexia, mechanistic support for obesity's benefit is lacking. Given that obesity and cachexia are both accompanied by metabolic dysregulation, we sought to investigate the impact of obesity on skeletal muscle mass loss and mitochondrial dysfunction in murine cancer cachexia. Male C57BL/6 mice were given a purified high fat or standard diet for 16weeks before being implanted with 106 Lewis lung carcinoma (LLC) cells. Mice were monitored for 25days, and hindlimb muscles were collected for cachexia indices and mitochondrial assessment via western blotting, high-resolution respirometry and transmission electron microscopy (TEM). Obese LLC mice experienced significant tumour-free body weight loss similar to lean (-12.8% vs. -11.8%, P=0.0001) but had reduced survival (33.3% vs. 6.67%, χ2 =10.04, P=0.0182). Obese LLC mice had reduced muscle weights (-24%, P<0.0354) and mCSA (-16%, P=0.0004) with similar activation of muscle p65 (P=0.0337), and p38 (P=0.0008). ADP-dependent coupled respiration was reduced in both Obese and Obese LLC muscle (-30%, P=0.0072) consistent with reductions in volitional cage activity (-39%, P<0.0001) and grip strength (-41%, P<0.0001). TEM revealed stepwise reductions in intermyofibrillar and subsarcolemmal mitochondrial size with Obese (IMF: -37%, P=0.0009; SS: -21%, P=0.0101) and LLC (IMF: -40%, P=0.0019; SS: -27%, P=0.0383) mice. Obese LLC mice had increased pAMPK (T172; P=0.0103) and reduced FIS1 (P=0.0029) and DRP1 (P<0.0001) mitochondrial fission proteins, which were each unchanged in Lean LLC. Further, mitochondrial TEM analysis revealed that Obese LLC mice had an accumulation of damaged and dysfunctional mitochondria (IMF: 357%, P=0.0395; SS: 138%, P=0.0174) in concert with an accumulation of p62 (P=0.0328) suggesting impaired autophagy and clearance of damaged mitochondria. Moreover, we observed increases in electron lucent vacuoles only in Obese LLC muscle (IMF: 421%, P=0.0260; SS: 392%, P=0.0192), further supporting an accumulation of damaged materials that cannot be properly cleared in the obese cachectic muscle. Taken together, these results demonstrate that obesity is not protective against cachexia and suggest exacerbated impairments to mitochondrial function and quality control with a particular disruption in the removal of damaged mitochondria. Our findings highlight the need for consideration of the severity of obesity and pre-existing metabolic conditions when determining the impact of weight status on cancer-induced cachexia and functional mitochondrial deficits.

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

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. 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. 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. 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.