[The Construction of ROR1 Targeting Chimeric Antigen Receptor Modified T Cells and Its Killing Effect for ROR1-positive Tumor Cells].

OBJECTIVE: To test the killing effect of type Ⅰ receptor tyrosine kinase-like orphan receptor (ROR1) chimeric antigen receptor T cell (CAR-T) on several ROR1-expressing tumor cells in vitro. METHODS: The CAR gene was designed and synthesized by constructing the lentiviral vector plasmid, and BamHⅠ/EcoRⅠ was used to identify the plasmid. The expression levels of ROR1 among a variety of tumor cell lines were compared using flow cytometry (FCM). The killing effect of CAR-T on positive cells was detected by FCM, the LDH assay and ELISA. RESULTS: The double enzyme digestion identified CAR gene was successfully constructed to the lentivirus vector plasmid. FCM detection showed that the efficiency of CAR-T infection was about 47.23%. Multiple tumor cells expressed ROR1 in varying degrees. The FCM and the LDH assay indicated that CAR-T specifically killed ROR1-positive tumor cells. On positive target cells, more interferonI-γ (FN-γ) could be released during the CAR-T killing process than control T (P<0.05). CONCLUSION: We successfully constructed ROR1 CAR-T. CAR-T can specifically kill ROR1-positive tumor cells and cause the release of large amounts of IFN-γ, providing an experimental basis for clinical application.

[REGgamma promotes malignant behaviors of lung cancer cells].

OBJECTIVE: To determine the expression of proteasome aotivator gamma (REGgamma) in human lung cancer tissues and cell lines and its association with malignant biological behaviors. METHODS: Immunohistochemistry (IHC) was used to detect the expression of REGgamma in lung cancer and normal lung tissues. The expressions of REGgamma in lung cancer cells and normal epithelial cells were determined by Western blot. The H1975 lung cancer stable cell lines with different levels of REGgamma expression were constructed and their proliferations were evaluated by MTT assay. PI staining was used to assess the influence of REGgamma on cell growth cycle. The effect of REGgamma on the migration of lung cancer cells were observed with the cell scratch experiment. RESULTS: Lung cancer tissues had significantly higher levels of REGgamma expression than normal tissues. Similarly, lung cancer cell lines showed higher levels of REGgamma expression than the normal epithelial cell line. The overexpression of REGgamma enhanced cancer cell proliferations (P < 0.05), promoted more cells into the S+G2/M phase (P < 0.05) and promoted the migration of cancer cells (P < 0.05). All of these effects were reversed after suppression of REGgamma. CONCLUSION: REGgamma facilitates malignant biological behaviors of lung cancers.

[Study on the anti-Lewis lung carcinoma effect of metformin combined with MCT1 inhibitor CHC].

OBJECTIVE: To investigate the antitumor effect of the combination of metformin with alpha-cyano-4-hydroxycinnamic acid (CHC, a MCT1 inhibitor) in the treatment of Lewis lung cancer. METHODS: In vitro, the utilization of lactate acid was measured by lactate assay in cultured medium and the inhibition of LL/2 cell proliferation of four groups [control group, metformin group (1 mmol/L and 5 mmol/L), CHC group (5 mmol/L) and the combination group (metformin 5 mmol/L and CHC 5 mmol/L)] was detected in 24 h, 48 h, and their apoptosis in 24 h was also detected. In vivo, twenty eight C57BL/6 mice bearing LL/2 (5 x 10(5)) subcutaneous Lewis lung cancer on the right flank was established and then randomly assigned into four groups: control, metformin (200 mg/kg body mass in 0.1 mL i. g. with NS 0.1 mL i. p.), CHC (100 mg/kg body mass in 0.1 mL i.p. with NS 0.1 mL i. g. ) and the combination (metformin 200 mg/kg body mass in 0.1 mL i.g. with CHC 100 mg/kg body mass in 0.1 mL i. p.). Tumor volume was measured. The pathologic observation and apoptotic analysis of tumors was assessed by TUNEL assay. RESULTS: Compared to the contorl, metformin or CHC alone, combination of two drugs leaded to a significant lactate acid production in cultured medium and the inhibition of LL/ 2 cell viability (P < 0.05). In vivo, the systemic administration of two drugs leaded to obvious retarded tumor growth compared with used alone in early stage (P < 0.05). The TUNEL assay showed the significantly increased number of apoptosis cells in tumor tissues from the combination group. CONCLUSION: Combination of metformin and CHC transformed the lactic acid metabolism in LL/2 cells and induced cell apoptosis and showed the antitumor effect.

Silencing of pkm2 increases the efficacy of docetaxel in human lung cancer xenografts in mice.

Tumor aerobic glycolysis, or the Warburg effect, plays important roles in tumor survival, growth, and metastasis. Pyruvate kinase isoenzyme M2 (PKM2) is a key enzyme that regulates aerobic glycolysis in tumor cells. Recent research has shown that PKM2 can be used as a tumor marker for diagnosis and, in particular, as a potential target for cancer therapy. We investigated the effects of combining shRNA targeting PKM2 and docetaxel on human A549 lung carcinoma cells both in vivo and in vitro. We observed that the shRNA can significantly downregulate the expression level of PKM2. The decrease of PKM2 resulted in a decrease in ATP synthesis, which caused intracellular accumulation of docetaxel. Furthermore, the combination of pshRNA-pkm2 and docetaxel inhibited tumor growth and promoted more cancer cell apoptosis both in vivo and in vitro. Our findings suggest that targeting tumor glycolysis can increase the efficacy of chemotherapy. In particular, the targeting of PKM2 could, to some extent, be a new way of reversing chemotherapy resistance to cancer therapy.

The Promotion of Erythropoiesis via the Regulation of Reactive Oxygen Species by Lactic Acid.

The simultaneous increases in blood lactic acid and erythrocytes after intense exercise could suggest a link between lactate and the erythropoiesis. However, the effects of lactic acid on erythropoiesis remain to be elucidated. Here, we utilized a mouse model to determine the role of lactic acid in this process in parallel with studies using leukaemic K562 cells. Treatment of K562 cells in vitro with lactic acid increased the mRNA and protein expression of haemoglobin genes and the frequency of GPA+ cells. Also, increases in haematocrit and CD71-/Ter119+ erythroid cells were observed in lactic acid-treated mice, which showed a physiological increase in blood lactate. Mouse bone marrow CD34+/CD117- cells showed an increase in erythroid burst-forming units after stimulation with lactic acid in vitro. Furthermore, lactic acid increased the intracellular reactive oxygen species (ROS) content in bone marrow and in K562 cells. Erythroid differentiation induced in Haematopoietic Stem Cells (HSCs) and K562 cells by lactic acid was abolished by reducing ROS levels with SOD or 2-mercaptoethanol, which suggests that ROS is a critical regulator of this process. These findings provide a better understanding of the role of lactic acid in cellular metabolism and physiological functions.

Intratumoral expression of mature human neutrophil peptide-1 potentiates the therapeutic effect of doxorubicin in a mouse 4T1 breast cancer model.

Human neutrophil peptides (HNPs) 1-3 possess a high degree of similarity and are deregulated in many types of human tumors. Previous studies have demonstrated that tumor cell lines and microdissected fresh tumor cells express HNP1-3. In vitro, HNP1-3 have been reported to be cytotoxic to various types of tumor cells. Previously, we observed that the intracellular expression of HNP1 increased plasma membrane permeability in A549 cells and inhibited in vivo tumor angiogenesis. Based on these findings, we inferred that the intratumoral expression of HNP1 may benefit chemotherapy in solid tumors. In the present study, we established a mouse 4T1 breast cancer model imitating locally advanced breast cancer (LABC) and we injected the mice intratumorally with plasmid HNP1 (pHNP1). Doxorubicin (Dox) was administered twice i.v. at 5 mg/kg body weight on day 1 and 8. The possible mechanisms were investigated by evaluating cell proliferation and apoptosis, intracellular Dox accumulation, mitochondrial transmembrane potential (ΔΨm) and ultrastructural alteration of intratumoral microvessels. Compared with the single agent treatment, the combination of Dox and pHNP1 resulted in a significant delay in in vivo tumor growth and a decrease in lung metastasis. This chemosensitization effect was also observed in an A549 lung cancer model treated with cisplatin (DDP) and pHNP1. MTT assay and Annexin V staining demonstrated that 4T1 cells pre-transfected with pHNP1 were significantly more sensitive to Dox, causing increased proliferation inhibition and apoptosis. Further investigations showed that the intracellular expression of HNP1 enhanced Dox accumulation and significantly impaired mitochondrial ΔΨm. Moreover, in tumor tissues, HNP1 mediated intratumoral microvessel normalization and caused significant in situ tumor cell apoptosis. Our study suggests that intratumoral expression of HNP1 can significantly improve the therapeutic efficacy of Dox in breast cancer, abrogate the influence of multidrug resistance and enhance medication safety. Our findings may be important for the clinical therapeutic strategies of cancer chemotherapy.

K­ras gene mutation as a predictor of cancer cell responsiveness to metformin.

An increasing number of studies support the use of metformin, a common antidiabetic drug, as a novel anticancer therapeutic. However, its mechanism of action has yet to be identified. In the current study, metformin was observed to effectively inhibit the growth of the K-ras mutant but not wild-type tumors in vivo. The antitumor effects of metformin were mediated by the induction of apoptosis and inhibition of proliferation in vivo. In addition, metformin induced apoptosis in the K-ras mutant tumors, A549 and PANC-1, but not in the K-ras wild-type tumor, A431, in vitro. Similarly, at lower concentrations, metformin inhibited cell proliferation in the K-ras mutant, but not in the K-ras wild-type tumor cells in vitro. These observations indicate that tumors with K-ras mutations are sensitive to metformin therapy. In addition, metformin significantly arrested K-ras mutant and wild-type tumor cells in G1 phase in vitro and metformin downregulated two important downstream effectors of the Ras signaling pathway in K-ras mutant tumors. Metformin was concluded to function as a potential K-ras-targeting agent that has potential for cancer therapy.

Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts.

Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p<0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy.