BACKGROUND: Hyperthermia (HT), while a cancer treatment approach, isn't always effective alone. Therefore, identifying hyperthermia enhancers is crucial. We demonstrated that Mito-TEMPO ([2-[(1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl) amino]-2-oxoethyl]-triphenylphosphanium, MT) acts as a potent thermosensitizer, promoting cell death in human cervical cancer (HeLa) cells. METHODS: Cells were pretreated with 0.4 mM MT for 5 minutes, followed by exposure to hyperthermia (42 °C for 60 minutes). The impacts of MT/HT on cell viability, proliferation, apoptosis, endoplasmic reticulum (ER) stress, apoptosis-related proteins and autophagy, autophagy-related proteins expression were measured. The relationships between autophagy and apoptosis were further investigated using the specific autophagy inhibitor chloroquine (CQ) and the autophagy inducer rapamycin (Rapa). RESULTS: The combined treatment reduced the mitochondrial membrane potential (MMP) and increased ROS production. It also upregulated the pro-apoptotic protein Bax and downregulated anti-apoptotic proteins such as Bcl-2 and MCL-1. As a result, Caspase-3 was activated. Additionally, the combined treatment upregulated the expression of p-PERK/PERK, ATF-4, CHOP proteins. Moreover, the combined treatment also increased the expression of LC3 II and p62, decreased expression of LAMP 1 and Cathepsin D and increased lysosomal pH, indicating coordinated changes in autophagy regulation. Notably, intensification of apoptosis induced by the combined treatment was observed with CQ, whereas attenuation was seen with Rapa. CONCLUSIONS MT effectively enhanced HT-induced apoptosis in HeLa cells. Elevated ER stress and interruption of autophagy flux are the possible underlying molecular mechanisms for this phenomenon. These findings suggested MT can act as a potential thermosensitizer, highlighting its versatility in cancer treatment strategies.
Humans ; Apoptosis/drug effects* ; Autophagy/drug effects* ; HeLa Cells ; Uterine Cervical Neoplasms/therapy* ; Female ; Hyperthermia, Induced ; Spin Labels ; Endoplasmic Reticulum Stress/drug effects* ; Cyclic N-Oxides/pharmacology* ; Cell Survival/drug effects*

OBJECTIVE: Acute lung injury (ALI) is a serious respiratory dysfunction caused by pathogen or physical invasion. The strong induced inflammation often causes death. Tanshinone IIA (Tan-IIA) is the major constituent of Salvia miltiorrhiza Bunge and has been shown to display anti-inflammatory effects. The aim of the current study was to investigate the effects of Tan-IIA on ALI. METHODS: A murine model of lipopolysaccharide (LPS)-induced ALI was used. The lungs and serum samples of mice were extracted at 3 days after treatment. ALI-induced inflammatory damages were confirmed from cytokine detections and histomorphology observations. Effects of Tan-IIA were investigated using in vivo and in vitro ALI models. Tan-IIA mechanisms were investigated by performing Western blot and flow cytometry experiments. A wound-healing assay was performed to confirm the Tan-IIA function. RESULTS: The cytokine storm induced by LPS treatment was detected at 3 days after LPS treatment, and alveolar epithelial damage and lymphocyte aggregation were observed. Tan-IIA treatment attenuated the LPS-induced inflammation and reduced the levels of inflammatory cytokines released not only by inhibiting neutrophils, but also by macrophage. Moreover, we found that macrophage activation and polarization after LPS treatment were abrogated after applying the Tan-IIA treatment. An in vitro assay also confirmed that including the Tan-IIA supplement increased the relative amount of the M2 subtype and decreased that of M1. Rebalanced macrophages and Tan-IIA inhibited activations of the nuclear factor-κB and hypoxia-inducible factor pathways. Including Tan-IIA and macrophages also improved alveolar epithelial repair by regulating macrophage polarization. CONCLUSION This study found that while an LPS-induced cytokine storm exacerbated ALI, including Tan-IIA could prevent ALI-induced inflammation and improve the alveolar epithelial repair, and do so by regulating macrophage polarization.
Abietanes ; Acute Lung Injury/drug therapy* ; Animals ; Cytokine Release Syndrome ; Cytokines ; Inflammation/drug therapy* ; Lipopolysaccharides/toxicity* ; Macrophage Activation ; Macrophages ; Mice ; Triacetoneamine-N-Oxyl/pharmacology*

OBJECTIVE: To investigate the effect of the chemoprotectant tempol on the anti-tumor activity of cisplatin (DDP). METHODS: The cellular toxicity of tempol in human colon cancer SW480 cells and mouse colon cancer CT26 cells were evaluated using MTT and cell counting kit-8 assays. CalcuSyn software analysis was used to determine the interaction between tempol and DDP in inhibition of the cell viability. A subcutaneous homograft mouse model of colon cancer was established. The mice were randomly divided into control group, tempol group, cisplatin group and tempol + DDP treatment group with intraperitoneal injections of the indicated agents. The tumor size, body weight and lifespan of the mice were measured, and HE staining was used to analyze the cytotoxic effect of the agents on the kidney and liver. Immunohistochemistry and Western blotting were performed to detect the expression of Bax and Bcl2 in the tumor tissue, and TUNEL staining was used to analyze the tumor cell apoptosis. The level of reactive oxygen species (ROS) in the tumor tissue was determined using flow cytometry. RESULTS: Tempol showed inhibitory effects on the viability of SW480 and CT26 cells. CalcuSyn software analysis showed that tempol had a synergistic anti-tumor effect with DDP (CI < 1). In the homograft mouse model, tempol treatment alone did not produce obvious anti-tumor effect. HE staining showed that the combined use of tempol and DDP alleviated DDP-induced fibrogenesis in the kidneys, but tempol also reduced the anti-tumor activity of DDP. Compared with the mice treated with DDP alone, the mice treated with both tempol and DDP had a significantly larger tumor size ( < 0.01) and a shorter lifespan ( < 0.05). Tempol significantly reversed DDP-induced expression of Bax and Bcl2 in the tumor tissue and tumor cell apoptosis ( < 0.001), and obviously reduced the elevation of ROS level in the tumor tissue induced by DDP treatment ( < 0.05). CONCLUSIONS Tempol can attenuate the anti-tumor effect of DDP while reducing the side effects of DDP. Caution must be taken and the risks and benefits should be carefully weighed when considering the use of tempol as an anti-oxidant to reduce the toxicities of DDP.
Animals ; Antineoplastic Agents ; Antioxidants ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cisplatin ; Cyclic N-Oxides ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Mice ; Spin Labels

Progressive cribriform and zosteriform hyperpigmentation (PCZH) is a distinctive pigmentary disorder observed along the lines of Blaschko. Clinically, the lesions appear as uniformly tan, cribriform macular hyperpigmentation with a zosteriform distribution, without a history of rash, injury, inflammation, or other associated cutaneous or internal abnormalities. Histopathological specimens show increased melanin pigmentation in the basal cell layer with a complete absence of nevus cells. We report 8 cases of PCZH and review the literature on this peculiar disorder.
Exanthema ; Hyperpigmentation* ; Inflammation ; Melanins ; Nevus ; Pigmentation ; Triacetoneamine-N-Oxyl

Tanshinone IIA (Tan IIA) is a pharmacologically active substance extracted from the rhizome of Salvia miltiorrhiza Bunge (also known as the Chinese herb Danshen), and is widely used to treat atherosclerosis. The pregnane X receptor (PXR) is a nuclear receptor that is a key regulator of xenobiotic and endobiotic detoxification. Tan IIA is an efficacious PXR agonist that has a potential protective effect on endothelial injuries induced by xenobiotics and endobiotics via PXR activation. Previously numerous studies have demonstrated the possible effects of Tan IIA on human umbilical vein endothelial cells, but the further mechanism for its exerts the protective effect is not well established. To study the protective effects of Tan IIA against hydrogen peroxide (H₂O₂) in human umbilical vein endothelial cells (HUVECs), we pretreated cells with or without different concentrations of Tan IIA for 24 h, then exposed the cells to 400 μM H₂O₂ for another 3 h. Therefore, our data strongly suggests that Tan IIA may lead to increased regeneration of glutathione (GSH) from the glutathione disulfide (GSSG) produced during the GSH peroxidase-catalyzed decomposition of H₂O₂ in HUVECs, and the PXR plays a significant role in this process. Tan IIA may also exert protective effects against H₂O₂-induced apoptosis through the mitochondrial apoptosis pathway associated with the participation of PXR. Tan IIA protected HUVECs from inflammatory mediators triggered by H₂O₂ via PXR activation. In conclusion, Tan IIA protected HUVECs against H₂O₂-induced cell injury through PXR-dependent mechanisms.
Apoptosis ; Asian Continental Ancestry Group ; Atherosclerosis ; Endothelial Cells* ; Glutathione ; Glutathione Disulfide ; Human Umbilical Vein Endothelial Cells ; Humans ; Hydrogen Peroxide ; Inflammation ; Oxidative Stress ; Regeneration ; Rhizome ; Salvia miltiorrhiza ; Triacetoneamine-N-Oxyl ; Xenobiotics

Two Whooper swan (Cygnus cygnus) died after suffering from pododermatitis, lethargy, and ataxia; necropsy was performed. Grossly, the liver was swollen and firm. The kidney and spleen were also enlarged and a pale tan color. On histopathologic examination with Congo red staining, amyloidosis was noted in liver, spleen, and kidney. In addition, marked osseous metaplasia was present in the liver. Based on these results, systemic amyloidosis involving liver, spleen, and kidney with osseous metaplasia in the liver was diagnosed. Study results indicate that an inflammatory reaction associated with pododermatitis had a role in the amyloidosis in this particular case.
Amyloidosis* ; Ataxia ; Congo Red ; Kidney ; Lethargy ; Liver ; Metaplasia ; Spleen ; Triacetoneamine-N-Oxyl

PURPOSE: This study is to investigate the effect of captopril when combined with irradiation. MATERIALS AND METHODS: 4T1 (mouse mammary carcinoma) cells were injected in the right hind leg of Balb/c mice. Mice were randomized to four groups; control (group 1), captopril-treated (group 2), irradiated (group 3), irradiated and captopril-treated concurrently (group 4). Captopril was administered by intraperitoneal injection (10 mg/kg) daily and irradiation was delivered on the tumor-bearing leg for 15 Gy in 3 fractions. Surface markers of splenic neutrophils (G-MDSCs) and intratumoral neutrophils (tumor-associated neutrophils [TANs]) were assessed using flow cytometry and expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 alpha (HIF-1α) of tumor was evaluated by immunohistochemical (IHC) staining. RESULTS: The mean tumor volumes (±standard error) at the 15th day after randomization were 1,382.0 (±201.2) mm³ (group 1), 559.9 (±67.8) mm³ (group 3), and 370.5 (± 48.1) mm³ (group 4), respectively. For G-MDSCs, irradiation reversed decreased expression of CD101 from tumor-bearing mice, and additional increase of CD101 expression was induced by captopril administration. Similar tendency was observed in TANs. The expression of tumor-necrosis factor-associated molecules, CD120 and CD137, are increased by irradiation in both G-MDSCs and TANs. Further increment was observed by captopril except CD120 in TANs. For IHC staining, VEGF and HIF-1α positivity in tumor cells were decreased when treated with captopril. CONCLUSION: Captopril is suggested to have additional effect when combined to irradiation in a murine tumor model by modulation of MDSCs and angiogenesis.
Angiotensin-Converting Enzyme Inhibitors ; Animals ; Captopril* ; Flow Cytometry ; Hypoxia-Inducible Factor 1 ; Injections, Intraperitoneal ; Leg ; Mice ; Neutrophils ; Radiotherapy ; Random Allocation ; Triacetoneamine-N-Oxyl ; Vascular Endothelial Growth Factor A

The present study was aimed to investigate the roles of renal sympathetic nerve and oxidative stress in the development of foot shock-induced hypertension. Ninety rats were divided into 6 groups (the number of each group was 15): control group, foot shock group, denervation of renal sympathetic nerve group, denervation of renal sympathetic nerve + foot shock group, Tempol treatment + foot shock group, denervation of renal sympathetic nerve + Tempol treatment + foot shock group. Rats were received electrical foot shock for 14 days (2-4 mA, 75 V, shocks of 50-100 ms every 30 s, for 4 h each session through an electrified grid floor every day). Renal sympathetic ablation was used to remove bilateral renal sympathetic nerve in rats (rats were allowed to recover for one week before the beginning of the foot shock procedure). The antioxidant Tempol was injected intraperitoneally at 1 h before foot shock. Systolic blood pressure was measured at 1 h after foot shock on day 0, 3, 7, 10 and 14. Contents of thiobarbituric acid reactive substance (TBARS), renin, angiotensin II (AngII) and glutathione peroxidase (GSH-Px) in plasma were measured by ELISA after 14-day foot shock. The results showed that systolic blood pressure of foot shock group was significantly increased (P < 0.05) compared with that of control group from day 7 to day 14 of foot shock. Denervation of renal sympathetic nerve and/or Tempol treatment significantly reduced the increase of systolic blood pressure induced by foot shock. Levels of TBARS, renin and AngII in plasma were increased significantly in foot shock group compared with that of control group (P < 0.05). Plasma GSH-Px concentration was decreased in foot shock group rats compared with that of control group (P < 0.05). Denervation of renal sympathetic nerve and/or tempol treatment significantly reduced the increase in TBARS, renin, AngII levels induced by foot shock in comparison with that of foot shock group (P < 0.05), but had no effects on the reduction of GSH-Px concentration. The results suggest that renal sympathetic nerve may play an important role in the development of foot shock-induced hypertension, and renal sympathetic nerve may influence oxidative stress and directly or indirectly activate renin-angiotensin-aldosterone system, so the foot shock-induced high blood pressure may be maintained and hypertension may therefore be produced.
Animals ; Antioxidants ; pharmacology ; Blood Pressure ; Cyclic N-Oxides ; pharmacology ; Denervation ; Electric Stimulation ; Hypertension ; physiopathology ; Kidney ; innervation ; Oxidative Stress ; Rats ; Renin-Angiotensin System ; Spin Labels ; Sympathetic Nervous System ; physiology

We report a spontaneous embryonal rhabdomyosarcoma in the abdominal cavity of an aged (88-week-old) Sprague-Dawley rat. The animal had a firm lobulated 5 x 5 x 4.5 cm mass in the abdominal cavity that was whitish to tan with necrotic and hemorrhagic plaques. Microscopically, the mass contained nodules with spindle or globoid shaped neoplastic cells with abundant eosinophilic cytoplasm and round or elongated nuclei mixed with other spindle cells with a filamentous appearance and scanty cytoplasm. Multinucleated cells and cross-striations were also observed. The neoplastic cells were positive for vimentin, desmin, and alpha-smooth muscle actin, especially the small spindle cells.
Abdominal Cavity* ; Actins ; Animals ; Cytoplasm ; Desmin ; Eosinophils ; Rats* ; Rats, Sprague-Dawley* ; Rhabdomyosarcoma, Embryonal* ; Triacetoneamine-N-Oxyl ; Vimentin

OBJECTIVETo explore the myeloid-derived suppressor cell (MDSC) expression in the peripheral blood and lesions of 4NQO-induced tongue carcinoma in mouse.

METHODSThe established 4NQO mouse model was used to analyze the distribution of MDSC and T cell subsets in the peripheral blood by flow cytometry. The relations of MDSC with T cell subsets and CD4⁺/CD8⁺ changes were evaluated. The distribution of MDSC in the lesions of tongues was analyzed by immu- nohistochemistry, and the expression of arginase 1 (ARG-1) in tongue tissues was detected by real-time polymerase chain reaction.

RESULTSDuring tumor progression, a significant increase was observed in the frequency of MDSC in the peripheral blood of 4NQO treated mice (P < 0.01). The frequency of MDSC was positively correlated with systemic CD3⁺CD8+T cells but negatively correlated with the CD4⁺/CD8⁺ ratio. Squamous cell carcinomas were extensively infiltrated with MDSC, whereas dysplastic area and normal tongue mucosa had only sparse MDSC infiltration. The majority of MDSCs were located in the stroma, particularly along the tumor invasive front. Moreover, 4NQO-treated mice showed significantly higher ARG-1 mRNA levels in the tumor site (P<0.01).

CONCLUSIONMDSC may contribute to oral tumor progression and represents a potential target for immunotherapy of oral cancer.

4-Nitroquinoline-1-oxide ; Animals ; Arginase ; Cell Count ; Flow Cytometry ; Mice ; Models, Animal ; Myeloid-Derived Suppressor Cells ; immunology ; Real-Time Polymerase Chain Reaction ; T-Lymphocyte Subsets ; immunology ; Tongue Neoplasms ; immunology