[Effects of normal mitochondrial transplantation on proliferation, apoptosis and stemness of triple-negative breast cancer cells].

Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group (P＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group (P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group (P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group (P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group (P＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group (P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group (P＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.

[Diagnostic values of conventional tumor markers and their combination with chest CT for patients with stageâ A lung cancer].

Objective: To investigate the diagnostic efficiency of conventional serum tumor markers and their combination with chest CT for stage ⅠA lung cancer. Methods: A total of 1 155 patients with stage ⅠA lung cancer and 200 patients with benign lung lesions (confirmed by surgery) treated at the Cancer Hospital, Chinese Academy of Medical Sciences from January 2016 to October 2020 were retrospectively enrolled in this study. Six conventional serum tumor markers [carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), squamous cell carcinoma associated antigen (SCCA), cytokeratin 19 fragment (CYFRA21-1), neuron-specific enolase (NSE), and gastrin-releasing peptide precursor (ProGRP)] and chest thin-slice CT were performed on all patients one month before surgery. Pathology was taken as the gold standard to analyze the difference of positivity rates of tumor markers between the lung cancer group and the benign group, the moderate/poor differentiation group and the well differentiation group, the adenocarcinoma group and the squamous cell carcinoma group, the lepidic and non-lepidic predominant adenocarcinoma groups, the solid nodule group and the subsolid nodule group based on thin-slice CT, and subgroups of ⅠA1 to ⅠA3 lung cancers. The diagnostic performance of tumor markers and tumor markers combined with chest CT was analyzed using the receiver operating characteristic curve. Results: The positivity rates of six serum tumor markers in the lung cancer group and the benign group were 2.32%-20.08% and 0-13.64%, respectively; only the SCCA positivity rate in the lung cancer group was higher than that in the benign group (10.81% and 0, P=0.022). There were no significant differences in the positivity rates of other serum tumor markers between the two groups (all P>0.05). The combined detection of six tumor markers showed that the positivity rate of the lung cancer group was higher than that of the benign group (40.93% and 18.18%, P=0.004), and the positivity rate of the adenocarcinoma group was lower than that of the squamous cell carcinoma group (35.66% and 47.41%, P=0.045). The positivity rates in the poorly differentiated group and moderately differentiated group were higher than that in the well differentiated group (46.48%, 43.75% and 22.73%, P=0.025). The positivity rate in the non-lepidic adenocarcinoma group was higher than that in lepidic adenocarcinoma group (39.51% and 21.74%, P=0.001). The positivity rate of subsolid nodules was lower than that of solid nodules (30.01% vs 58.71%, P=0.038), and the positivity rates of stageⅠA1, ⅠA2 and ⅠA3 lung cancers were 33.33%, 48.96% and 69.23%, respectively, showing an increasing trend (P=0.005). The sensitivity and specificity of the combined detection of six tumor markers in the diagnosis of stage ⅠA lung cancer were 74.00% and 56.30%, respectively, and the area under the curve (AUC) was 0.541. The sensitivity and specificity of the combined detection of six serum tumor markers with CT in the diagnosis of stage ⅠA lung cancer were 83.0% and 78.3%, respectively, and the AUC was 0.721. Conclusions: For stage ⅠA lung cancer, the positivity rates of commonly used clinical tumor markers are generally low. The combined detection of six markers can increase the positivity rate. The positivity rate of markers tends to be higher in poorly differentiated lung cancer, squamous cell carcinoma, or solid nodules. Tumor markers combined with thin-slice CT showed limited improvement in diagnostic efficiency for early lung cancer.

[Treatment of liver cancer in vitro and in mice by monoclonal antibody targeting epithelial specific antigen-positive liver cancer stem cells in combination with cisplatin].

OBJECTIVE: To investigate the biological characteristics of monoclonal antibodies against human liver cancer stem cells and its therapeutic effect in combination with cisplatin in the treatment of hepatocellular carcinoma. METHODS: Cell culture in serum-free medium and PKH26 staining were used to determine the existence of cancer stem cells in human liver Bel7402-V3 cell line. The co-expression of antigen recognized by monoclonal antibody (McAb) 15D2 and epithelial specific antigen (ESA) and PKH26-positive cells in the Bel7402-V3 cells were detected by immunofluorescence assay. Serum-free suspension culture was used to detect the self-renewal ability of 15D2-positive Bel7402-V3 cells sorted by flow cytometry and the effect of 15D2 on the self-renewal ability of Bel7402-V3 cells. The effect of 15D2 on cisplatin resistance in the cells was examined by CCK8 method. The inhibitory effect of 15D2 combined with cisplatin on the transplanted tumor growth in mice was also observed. RESULTS: Single PKH26-positive cells were observed in the Bel7402-V3 cell spheroids cultured for 11 days. Immunofluorescence assay showed that the 15D2-recognized antigen could be conjugated with PKH26 and ESA and co-localized on Bel7402-V3 cells. The spheroid formation rate of 15D2-positive cells in serum-free medium was significantly higher than that of 15D2-negative cells [(30.4±3.4)% vs. (8.8±1.8)%, P<0.01]. The cisplatin resistance of 15D2-positive cells was obviously higher than that of 15D2-negative cells (IC50: 1.014 µmol/L vs. 0.365 µmol/L). McAb 15D2 significantly suppressed the spheroid formation of Bel7402-V3 cells, with an inhibition rate of 37.5%. McAb 15D2 also notably inhibited the cisplatin resistance of Bel7302-V3 cells. The IC50 was 0.211 µg/ml in the 15D2 group and 0.325 µg/ml in the control group. The mouse experiment showed that the tumor growth rates of 50 mg/kg, 25 mg/kg and 12.5 mg/kg 15D2-treatment groups were 82.6%, 71.4% and 60.0%, respectively; that of the 50 mg/kg 15D2 + cisplatin group was 91.0%, and that of the cisplatin monotherapy was 56.7%. CONCLUSION: McAb 15D2 is a functional monoclonal antibody targeting liver cancer stem cells, which could be a potential monoclonal antibody drug for the stem cell-targeted therapy of liver cancer.

A platelet function inhibitor purified from Vipera russelli siamensis (Smith) snake venom.

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75, a potent platelet function inhibitor was purified from Vipera russelli siamensis venom. It appeared as a single protein band on polyacrylamide gel electrophoresis in the presence or absence of SDS, and consists of 123 amino acid residues. Its NH2-terminal residue is serine. It showed the following characteristics: molecular weight, 13,800; isoelectric point, 10.4; LD50, 0.5 +/- 0.12 mg/kg (i.v.). The platelet inhibitor exhibited phospholipase A2 activity with a specific activity of 35 mumoles/min/mg. From 2 g of the venom, 70 mg of the purified inhibitor was obtained. Inhibition of human platelet aggregation induced by ADP or adrenaline was dose-dependent, with ID50 of 1.14 micrograms/ml or 0.37 microgram/ml, respectively. The platelet aggregation induced by thrombin or collagen was also inhibited and the inhibitory activity on platelet aggregation was heat stable (at 100 degrees C, 20 min) in an acidic medium (pH 5.8), while its phospholipase A2 activity was relatively heat labile under the same condition. The release of 3H-serotonin in platelets stimulated by ADP was also inhibited and this was positively correlated with inhibition of platelet aggregation induced by ADP (r = 0.998, P less than 0.002).

Biochemical characterization of two hemorrhagic proteases from the venom of Lachesis muta (bushmaster).

Two hemorrhagic proteases, Lachesis hemorrhagic toxins a and b (LHTa and LHTb), were isolated from the venom of Lachesis muta, which is distributed in Central and South America. One protease showed strong hemorrhagic action, while the other showed weak hemorrhagic activity even though the two enzymes are very similar in their chemical properties. Neither enzyme hydrolyzed arginine esters, but both hydrolyzed casein and reduced fibrinogen. The A alpha chain of fibrinogen was hydrolyzed first, and the B beta chain was hydrolyzed later. The gamma chain of fibrinogen was resistant to hydrolysis. The molecular weights of LHTa and LHTb were very similar, 22,000 and 23,000, respectively. The amino acid composition of LHTa was also similar to that of LHTb. The secondary structure of LHTa as determined by Lippert's equation was 52% alpha helix, 17% beta sheet, and 31% random coil; that of LHTb was 47% alpha helix, 13% beta sheet, and 40% random coil.