Role of thioredoxin nitration in doxorubicin-induced apoptosis of neonatal rat cardiomyocytes

AIM：To investigate the role of thioredoxin nitration in the apoptosis of neonatal rat cardiomyocytes (NRCMs) induced by doxorubicin (DOX). METHODS：Cardiomyocytes treated with DOX were isolated from newborn Sprague-Dawley rats and cultured in vitro. NRCMs were treated with DOX alone (DOX group), pretreated with Mn (III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP), a peroxynitrite (ONOO-) scavenger, and then treated with DOX (MnTMPyP+DOX group), or treated with MnTMPyP alone (MnTMPyP group). NRCMs without any treatment served as a normal control (control group). The viability of the cells was examined by MTT assay, and the apoptosis was measured by Hoechst 33258 nuclear staining kit. The activity of caspase-3 was detected by spectrophotometry. The expression of cleaved poly(ADP-ribose) polymerase 1 (PARP-1), apoptosis signal-regulating kinase 1 (ASK1), phosphorylated ASK1 (p-ASK1), p38 mitogen-activated protein kinase (p38 MAPK) and phosphorylated p38 MAPK (p-p38 MAPK) was measured by Western blotting. Immunoprecipitation and immunoblotting were performed to detect the formation of Trx-ASK1 and Trx-nitrotyrosine. RESULTS：DOX induced significant apoptosis of NRCMs. MnTMPyP could significantly attenuate the apoptosis induced by DOX. Compared with control group, Trx nitration in DOX group increased obviously. The increases in activity of caspase-3 and expression of cleaved PARP-1 and p-p38 MAPK were also observed, besides the expression of Trx-ASK1 compound and p-ASK1 decreased significantly (P<005). MnTMPyP could decrease the nitration of Trx. The decreases in activity of caspase-3 and expression of cleaved PARP-1 and p-p38 MAPK were detected in MnTMPyP+DOX group, while the expression of Trx-ASK1 compound and p-ASK1 increased significantly (P<005). CONCLUSION: DOX could induce significant apoptosis of NRCMs and increase Trx nitration. The process was significantly attenuated by pretreatment with MnTMPyP. Therefore, Trx nitration may play an important role in doxorubicin-induced apoptosis of cardiomyocytes.     

Yellow wine polyphenol compounds attenuate doxorubicin-induced cardiotoxicity

AIM:To investigate the effect of yellow wine polyphenol compounds (YWPC) on doxorubicin (DOX)-induced cardiotoxicity and its mechanism. METHODS:Adult male SD rats (n=40) were randomly assigned to 4 groups, including control group, YWPC group, DOX group and YWPC+DOX group. After 4 weeks of intervention, the rats were anaesthetized to measure the cardiac function using echocardiogram. The pathological changes of the heart tissues in each group were observed. The methods of colorimetry, DHE staining, TUNEL staining and Western blot were used to investigate the levels of myocardial enzymes, oxidative stress and apoptosis. RESULTS:Compared with control group, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were significantly decreased, while left ventricular internal dimension at systole (LVIDs) and left ventricular internal dimension at diastole (LVIDd) were significantly decreased in the DOX group, which were notably alleviated in the DOX+YWPC group (P<0.05). In addition, the serum levels of lactate dehydrogenase, creatine kinase, aspartate aminotransferase and cardiac troponin I were significantly increased in DOX group as compared with control group. However, the levels of the above indicators were decreased in DOX+YWPC as compared with DOX group (P<0.05). Compared with control group, no significant pathological change was observed in YWPC group; however, damaged myocardial cells, nuclear lysis and disassembled muscle fiber were observed in DOX group, which were significantly alleviated in DOX+YWPC group. The levels of reactive oxygen species (ROS) production, superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were dramatically elevated, while the level of malondialdehyde (MDA) was decreased in DOX group as compared with control group. The levels of the above-mentioned indicators were significantly reversed in DOX+YWPC group as compared with DOX group (P<0.05). Meanwhile, the apoptotic rates of cardiomyocytes, as well as the levels of Bax/Bcl-2 ratio and cleaved caspase-3 were significantly increased in DOX group as compared with control group, which were notably alleviated in DOX+YWPC group (P<0.05). CONCLUSION:YWPC alleviates DOX-induced cardiotoxicity by reducing oxidative stress and inhibiting myocardial apoptosis.     

Effects of fructose-1,6-diphosphate on adriamycin-induced calcium and sarcoplosnic reticulum Ca2+-ATPase activity in cardiomyocytes of rats

AIM: To study the effects of fructose-1,6-diphosphate (FDP) on adriamycin (ADR)-induced calcium and sarcoplasmic reticulum Ca²⁺-ATPase activity in cardiomyocytes of rats. METHODS: Rats were treated with ADR by intraperitoneal injection (2.5 mg·kg^-1 body weight) once every two days for 11 days, and then ADR-treated rats were intervened by FDP at different dosages (ip) once every other day for 41 days. Enzyme linked immune absorption assay (ELISA) was employed to detect froponin I (CTnI). CK-MB was examined by monoclonal antibody. Intracellular free calcium concentration was measured on fluorescent spectrophotometry and SRCa²⁺-ATPase activity was examined by inorganic phosphate. RESULTS: FDP (300, 600, 1 200 mg·kg^-1) significantly reduced the levels of CTnI and CK-MB in serum. Decreased calcium and increased SRCa²⁺-ATPase activity in cardiomyocytes were also observed when ADR-treated rats were intervened by FDP (P＜0.01). CONCLUSION: FDP reduced the injury of cardiotoxicity induced by ADR via decreasing intracellular free calcium and increasing SRCa²⁺-ATPase activity in cardiomyocytes.     

Effects of c-Met on proliferation of triple negative breast cancer and sensitivity to doxorubicin

AIM: To investigate the effects of c-Met on the proliferation and the sensitivity to chemotherapeutic drugs of triple negative breast cancer cells. METHODS: Doxorubicin-resistant cells (MDA-MB-231/ADR) were established. The expression of c-Met at mRNA and protein levels in the MDA-MB-231/ADR cells and parental MDA-MB-231 cells was detected by real-time PCR and Western blotting. c-Met siRNA and plasmid or AKT siRNA were transfected into the cancer cells. The cell proliferation and the sensitivity to doxorubicin were determined by MTT assay. RESULTS: The expression of c-Met at mRNA and protein levels in MDA-MB-231/ADR cells was significantly higher than that in parental MDA-MB-231 cells. Transfection with pBABE-puro TPR-MET plasmid into the MDA-MB-231 cells induced cell proliferation and resistance to doxorubicin. Meanwhile, inhibition of c-Met in the MDA-MB-231/ADR cells by siRNA reversed the doxorubicin-resistance. In addition, over-expression of c-Met led to higher phosphorylation level of AKT, which was involved in the effects of c-Met on the MDA-MB-231 cell proliferation and doxorubicin-resistance. CONCLUSION: c-Met may have the potential as a therapeutic target in the treatment of triple negative breast cancer.     

Tanshinone ⅡA attenuates doxorubicin-induced injury in H9c2 cardiomyocytes via AMPK-mediated autophagy

AIM: To investigate the effect of tanshinone ⅡA on doxorubicin-induced rat H9c2 cardiomyocyte injury. METHODS: The H9c2 cardiomyocytes were treated with tanshinone ⅡA and/or doxorubicin with or without AMPK inhibitor dorsomorphin. The cell viability was measured by CCK-8 assay, the LDH release was examined for evaluating the cell injury, autophagy was analyzed by immunofluorescence staining, and AMPK activation was determined by Western blot. RESULTS: Compared with control group, the viability of H9c2 cells was decreased, the release of LDH was increased, the autophagy degree was increased, and AMPK activation was inhibited after treatment with doxorubicin (P<0.05). Compared with doxorubicin group, the treatment with tanshinone ⅡA restored the cell viability, reduced the release of LDH, further increased autophagy degree, and activated AMPK in the H9c2 cells (P<0.05). AMPK inhibitor dorsomorphin attenuated the abilities of tanshinone ⅡA to restore the cell viability, reduce the release of LDH, and increase autophagy degree in the H9c2 cells (P<0.05). CONCLUSION: Tanshinone ⅡA attenuates doxorubicin-induced injury in H9c2 cardiomyocytes, and its mechanism might be related to AMPK-mediated autophagy, which provides experimental and theoretical basis for the clinical application of tanshinone ⅡA in the prevention and treatment of doxorubicin-induced cardiomyocyte injury.     

Tanshinone IIA inhibits doxorubicin resistance in gastric cancer cells

AIM: To investigate the inhibitory effect and the specific mechanism of tanshinone IIA on doxorubicin (DOX)-resistant gastric cancer cells. METHODS: The sensitivity of gastric cancer cells lines to DOX was determined by MTT assay. DOX-resistant gastric cancer cell lines were established by step selection with increasing concentrations of DOX. The cell cycle arrest, apoptosis and autophagy related-markers were analyzed by flow cytometry and Western blot. The expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multi-drug resistance-associated protein 1 (MRP-1) was determined by RT-qPCR and Western blot. RESULTS: DOX-sensitive cell lines SNU-719 and SNU-601 as well as the cell lines relatively resistant to DOX including SNU-638, SNU-668, SNU-216 and SNU-620 were identified according to the IC₅₀ values of DOX for different cell lines. Two DOX-resistant cell lines SNU-719R and SNU-601R were also established. Tanshinone IIA inhibited the expression of MRP-1 in DOX-resistant cell lines. Compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in cancer cells decreased the G₂/M phase cell number, increased the protein expression of p21, decreased the protein expressions of cyclin B1 and cyclin-dependent kinase 1 (CDK1) in the SNU-719 R cells and SNU-620 cells. In addition, compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in the cancer cells increased the protein expressions of p53, Bax and LC3B-II, decreased the protein expression of Bcl-2 and p62 (P<0.05). CONCLUSION: Tanshinone IIA is an effective drug in the inhibition of DOX resistance in gastric cancer.     

Study of apoptotic thershold of cis-diamminedichloroplatinum and adriamycin on hepatocellular carcinoma

AIM:To study the thershold of hepatocellular carcinoma (HCC) apoptosis induced by adriamycin (ADM) and cis-diamminedichloroplatinum(CDDP). METHODS: Using primary human hepatocellular carcinoma culture, immunofluorescence staining of Hoechst 33 258 in HCC cells,and flow cytometric assay. RESULTS:24 h after HCC cells were cultured with ADM or CDDP, it were found there were dispersive fluorescences in normal cells nuclei, and compact particulate fluorescences in apoptosis cells nuclei by immunofluorescence st AIMing of Hoechst 33 258. The rate of HCC cells apoptosis was dependent on doses of ADM and CDDP. HCC cells apoptotic threshold were determined, i.e.ADM was 1.0 μg/mL and CDDP was 1.5μg/mL (The clinical apoptotic sensitive dosages were ADM 20 mg/m² and CDDP 30 mg/m². CONCLUSION: HCC cells apototic threshold of ADM and CDDP were efficient in clinical chemotherapy.     

Combination of Treg inhibition and intratumoral transfection of Ad.GM-CSF enhances the chemotherapeutic effect of doxorubicin against hepatocellular carcinoma

AIM: To explore the possibility that combination of regulatory T-cell (Treg) inhibition and intratumoral transfection of Ad.GM-CSF enhances the chemotherapeutic effect of doxorubicin against hepatocellular carcinoma (HCC). METHODS: C57BL/6J subcutaneous HCC model was established. The mice were randomly divided into 6 groups with 12 mice in each group when the tumor volume reached to 100-150 mm³: control group, cyclophosphamide(CTX) group, doxorubicin (DOX) group, Ad.GM-CSF group, CTX+DOX group and CTX+DOX+Ad.GM-CSF group. Four mice in each group were sacrificed for spleen cytotoxic T-cell(CTL) activity assay and tumor tissue histological examination 5 d after the final therapy. Eight mice in each group were examined for tumor growth and survival. RESULTS: CTX enhanced the chemotherapeutic effect of doxorubicin against HCC by inhibiting the tumor growth (P<0.05) and prolonging the mice survival (62.13 d±4.21 d vs 79.88 d±9.00 d, P<0.05), which was significantly strengthened by the combined use of Ad.GM-CSF (79.88 d±9.00 d vs 106.13 d±5.23 d, P<0.01). Compared with other groups, more tumor necrosis in tumor specimens and more infiltration of CD8⁺ T-lymphocytes in CTX+DOX+Ad.GM-CSF group were observed. Furthermore, the spleen cytotoxic T-cell(CTL) activity was significantly improved (P<0.05). CONCLUSION: Doxorubicin-based chemotherapy and immunotherapy by the method of Treg inhibition combined with intratumoral transfection of Ad.GM-CSF have synergistic effect against HCC.     

Effects of sucking porcine liver extracts and doxorubicin on BEL-7402 hepatoma cells transplanted in renal capsule of BALB/C nude mice

AIM: To investigate the effect of sucking porcine liver extracts (LE) and doxorubicin (DXR) on BEL-7402 hepatoma cells in the renal capsulae of BALB/c nude mice. METHODS: Histo-cellular morphology, mitotic counting, ultrastructural observation and in situ DNA labeling apoptotic detection were performed. RESULTS: Both LE and DXR can apparently inhibit the tumors' growth and induce the apotosis of hepatoma cells. LE exerted no apparent effect on the hepatoma cell mitosis, but DXR inhibited it. Electron-microscopic observations showed LE can induce the hepatoma cells to apoptosis. CONCLUSIONS: LE can induce the hepatoma cells to apoptosis, but has no apparent effect of their differentiation and proliferation. DXR can not only induce the hepatoma cells to apoptosis and inhibit their growth, but also can promote their differentiation.     

Doxorubicin induces stemness of mouse TNBC 4T1 cells through Stat3-Oct-4/CD44 signaling pathway

AIM:To investigate the stemness of mouse triple-negative breast cancer (TNBC) 4T1 cells induced by doxorubicin (DOX) and the underlying mechanism. METHODS:The 4T1 cells and MDA-MB-468 cells were treated with DOX at different concentrations (0, 0.05, 0.1 and 0.5 μmol/L) for 24 h, and the shape and viability of the cells were observed. The concentration of DOX at 0.1 μmol/L was chosen as the optimal concentration for the following experiments. The 4T1 cells and MDA-MB-468 cells resistant to DOX were established by continuous stimulation with DOX for 4 weeks, and named as 4T1-DOX and MDA-MB-468-DOX. Sphere formation assay was used to detect the stemness of 4T1 cells and MDA-MB-468 cells. The expression of CD133 was observed by immunofluorescence staining. The expression of CD44 was analyzed by flow cytometry. The protein levels of Stat3, phosphorylated Stat3 (p-Stat3) and Oct-4 were determined by Western blot. RESULTS:The sphere formation ability of the 4T1-DOX cells was stronger than that of the 4T1 control cells. The 4T1-DOX cells expressed high levels of the stemness markers CD133 and CD44 as compared with the 4T1 cells (P<0.05). Furthermore, the 4T1-DOX cells exhibited enhanced activation of Stat3 (p-Stat3) and increased expression of Oct-4 (P<0.05), while the expression of total Stat3 had no obvious variation. In addition, when activation of Stat3 was inhibited by WP1066, the protein levels of p-Stat3, Oct-4 and CD44 were down-regulated (P<0.05). Furthermore, inhibition of Stat3 phosphorylation reduced the sphere formation ability of the 4T1-DOX cells (P<0.05). CONCLUSION:DOX induces the stemness of mouse TNBC 4T1 cells through Stat3-Oct-4 signaling pathway.