Disheveled3 enhanced EMT and cancer stem-like cells properties via Wnt/ß-catenin/c-Myc/SOX2 pathway in colorectal cancer.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) and cancer stem-like cells (CSLCs) play crucial role in tumor metastasis and drug-resistance. Disheveled3 (DVL3) is involved in malignant behaviors of cancer. However, the role and potential mechanism of DVL3 remain elusive in EMT and CSLCs of colorectal cancer (CRC). METHODS: UALCAN and PrognoScan databases were employed to evaluate DVL3 expression in CRC tissues and its correlation with CRC prognosis, respectively. Transwell, sphere formation and CCK8 assay were used to assess metastasis, stemness and drug sensitivity of CRC cells, respectively. Western blotting and dual luciferase assay were performed to analyze the protein expression and Wnt/ß-catenin activation, respectively. Lentiviral transfection was used to construct the stable cell lines. Animal studies were performed to analyze the effect of silencing DVL3 on tumorigenicity and metastasis of CRC cells in vivo. RESULTS: DVL3 was overexpressed in CRC tissues and several CRC cell lines. DVL3 expression was also higher in CRC tissues with lymph node metastasis than tumor tissues without metastasis, and correlated with poor prognosis of CRC patients. DVL3 positively regulated the abilities of migration, invasion and EMT-like molecular changes in CRC cells. Moreover, DVL3 promoted CSLCs properties and multidrug resistance. We further identified that Wnt/ß-catenin was crucial for DVL3-mediated EMT, stemness and SOX2 expression, while silencing SOX2 inhibited DVL3-mediated EMT and stemness. Furthermore, c-Myc, a direct target gene of Wnt/ß-catenin, was required for SOX2 expression and strengthened EMT and stemness via SOX2 in CRC cells. Finally, knockdown of DVL3 suppressed tumorigenicity and lung metastasis of CRC cells in nude mice. CONCLUSION: DVL3 promoted EMT and CSLCs properties of CRC via Wnt/ß-catenin/c-Myc/SOX2 axis, providing a new strategy for successful CRC treatment.

TRDMT1 exhibited protective effects against LPS-induced inflammation in rats through TLR4-NF-κB/MAPK-TNF-α pathway.

BACKGROUND: Inflammation is a complex physiological and pathological process. Although many types of inflammation are well characterized, their physiological functions are largely unknown. tRNA aspartic acid methyltransferase 1 (TRDMT1) has been implicated as a stress-related protein, but its intrinsic biological role is unclear. METHODS: We constructed a Trdmt1 knockout rat and adopted the LPS-induced sepsis model. Survival curve, histopathological examination, expression of inflammatory factors, and protein level of TLR4 pathway were analyzed. RESULTS: Trdmt1 deletion had no obvious impact on development and growth. Trdmt1 deletion slightly increased the mortality during aging. Our data showed that Trdmt1 strongly responded in LPS-treated rats, and Trdmt1 knockout rats were vulnerable to LPS treatment with declined survival rate. We also observed more aggravated tissue damage and more cumulative functional cell degeneration in LPS-treated knockout rats compared with control rats. Further studies showed upregulated TNF-α level in liver, spleen, lung, and serum tissues, which may be explained by enhanced p65 and p38 phosphorylation. CONCLUSIONS: Our data demonstrated that Trdmt1 plays a protective role in inflammation by regulating the TLR4-NF-κB/MAPK-TNF-α pathway. This work provides useful information to understand the TRDMT1 function in inflammation.

Does static electric field from ultra-high voltage direct-current transmission lines affect male reproductive capacity? Evidence from a laboratory study on male mice.

With the development of ultra-high-voltage direct-current (UHVDC) transmission technology and increase in transmission voltage, the issue of environmental static electric field (SEF) pollution is standing out and its possible health effects have caused much public attention. In this study, the effects of chronic exposure to SEF on reproductive capacity of male mice were investigated. Twenty Institute of Cancer Research (ICR) mice were exposed to SEF (56.3 ± 1.4 kV/m, 49 days) generated by a high-voltage device. Several biological end points related to spermatogenesis and testicular function were evaluated, including reproductive organ coefficients, sperm motility and morphology, serum testosterone level, and testicular histology. No significant differences were found between the SEF-exposed and sham-exposed groups at the end of the exposure period. However, further observation through transmission electron microscopy revealed cristae losses in mitochondria of spermatogenic cells after SEF exposure. Nevertheless, the mitochondria injury did not affect sperm motility, which might be explained from the perspective of energy supply. That is, most of the energy required for sperm movement is generated by glycolysis which occurs in the cytoplasm rather than oxidative phosphorylation which occurs in mitochondria. In conclusion, this study indicates that exposure to SEF (56.3 ± 1.4 kV/m, 49 days) has limited effects on male reproductive capacity.

Molecular cloning and characterization of kiss1 in Brandt's voles (Lasiopodomys brandtii).

Kisspeptin, encoded by kiss1, has been regarded as a major modulator of mammalian puberty and fertility due to its stimulation on GnRH. Brandt's vole is one of the main pest species on the Inner Mongolian steppes for its striking reproductive capacity and kiss1 is a key candidate gene related to reproductive regulatory cascades. In this study, kiss1 cDNA was cloned from the hypothalamus of Brandt's voles and kiss1 mRNA levels were investigated in different tissues, and at different developmental stages, using high-throughput real-time PCR. The full-length kiss1 cDNA was 682bp, containing an ORF of 405bp, encoding 134 amino acids with a conserved kisspeptin-10 region. Kiss1 mRNA was specifically expressed in ovary, testicle, small intestine, kidney, liver and hypothalamus tissues, and was undetectable in other tissues, including pituitary, heart, adrenal gland, bladder and uterus. Sexual organs of both male and female voles enter a period of rapid development in the postnatal 4weeks and reach or approach sexual maturity by 8weeks after birth. Kiss1 mRNA levels in the hypothalamus did not show a significant difference between week 2 and week 4, indicating kiss1 mRNA levels may not be related to the rapid growth of the sexual organs in early developmental stages. Kiss1 transcripts significantly increased in both sexes 8weeks after birth, and then were maintained at high levels in adults, indicating its possible role in the onset of puberty and maintaining of reproductive activity. These results are helpful to further the study of kiss1 function in reproductive regulation of Brandt's voles.

ROR1 is a novel prognostic biomarker in patients with lung adenocarcinoma.

Currently, there is no reliable biomarker to clinically predict the prognosis of lung adenocarcinoma (ADC). The receptor-tyrosine-kinase like orphan receptor 1 (ROR1) is reported to be overexpressed and associated with poor prognosis in several tumors. This study aimed to examine the expression of ROR1 and evaluate its prognostic significance in human lung ADC patients. In this present study, Western blot analysis and immunohistochemistry were performed to characterize expression of ROR1 protein in lung ADC patients. The results revealed that ROR1 protein expression was significantly higher in lung ADC tissues than that in their adjacent non-tumor tissues. Patients at advanced stages and those with positive lymph node metastasis expressed higher level of ROR1 (P < 0.001). Moreover, Chi-square test showed that ROR1 expression was correlated to gender (P = 0.028), the 7th edition of the American Joint Committee on Cancer tumor-node-metastasis (AJCC TNM) staging system and lymph node metastasis (P < 0.001). Kaplan-Meier survival analysis indicated an association of high ROR1 expression with worse overall survival (OS) in lung ADC patients (P < 0.001). Multivariate COX regression analysis further confirmed that ROR1 is an independent prognostic predictor (P < 0.001, HR = 4.114, 95% CI: 2.513-6.375) for OS. Therefore, ROR1 expression significantly correlates with malignant attributes of lung ADC and it may serve as a novel prognostic marker in lung ADC patients.

Improving Antitumor Activity with N-Trimethyl Chitosan Entrapping Camptothecin in Colon Cancer and Lung Cancer.

Camptothecin (CPT) exerts very strong antitumor activities by suppressing the activity of DNA topoisomerase I, but its application is greatly limited owing to its low solubility and the instability of the active lactone form. To overcome this bottleneck, we prepared the novel camptothecin nanocolloids based on N-trimethyl chitosan (CPT-TMC) to efficiently administer CPT systemically. In this study, we investigated the antitumor activity of CPT-TMC against both colon cancer and lung cancer. In vitro cell experiments both CPT and CPT-TMC significantly inhibited the growth of CT26 cells and LL/2 cells, but no statistical difference was observed between CPT-TMC and CPT. In vivo studies, CPT-TMC more effectively inhibited tumor growth and prolonged survival time than CPT both in the CT26 colon carcinoma subcutaneous model and in the LL/2 Lewis lung carcinoma subcutaneous model. In addition, results of PCNA and CD31 immunohistochemical staining of tumor tissues also confirmed the improved antitumor effect of CPT-TMC. These findings suggest that N-trimethyl chitosan could increase the antitumor effect of CPT. Consequently, CPT delivery by N-trimethyl chitosan is a potential approach for effective treatment of cancer.

Synergistic antitumor effects of combined deguelin and cisplatin treatment in gastric cancer cells.

Deguelin is a naturally occurring rotenoid with marked chemopreventive and antitumor activity. Cisplatin, characterized by damaging DNA, is widely used in the chemotherapy of malignancies, including gastric cancer. The present study investigated whether synergistic effects exist between the combination of deguelin and cisplatin, and the possible mechanism in vitro. The interactive effects of deguelin in combination with various concentrations of cisplatin were evaluated in the human gastric cancer MGC-803 cell line. The inhibition of cell proliferation was determined by 3-(4,5)-dimethylthiazol(-2-yl)-2,5-diphenyltetrazolium bromide assay and the mechanisms underlying the effects were further evaluated by western blot analysis. The results revealed that the combined treatment of deguelin and cisplatin exhibited a marked inhibition of MGC-803 cell proliferation when compared with that of the single therapies in vitro. In addition, isobologram analysis revealed that this combined treatment showed a synergistic effect. These observations may have promising therapeutic value for gastric cancer and thus warrant further investigation.

Prognostic role of lemur tyrosine kinase 3 in postoperative gastric cancer.

The treatment of gastric cancer has been unsatisfactory thus far; therefore, novel targets and treatment strategies are urgently required. Lemur tyrosine kinase (LMTK)3 is an estrogen receptor-α (ERα) modulator with a central role in endocrine resistance in breast cancer. Moreover, the expression and polymorphisms of LMTK3 are correlated with the prognosis of breast cancer patients. Since estrogen receptor (ER) is also expressed and plays a role in gastric cancer, we herein investigated the expression of the LMTK3 protein in 83 gastric cancer patients by tissue microarray and analyzed the correlation between LMTK3 expression and the prognosis of gastric cancer. Our results demonstrated that LMTK3 was more frequently expressed in gastric cancer tissues compared to non-cancerous mucosa (79.5 vs. 45.8%, respectively; P=0.000). The LMTK3 expression was significantly correlated with the depth of invasion (P=0.002) and disease stage (P=0.035). The Kaplan-Meier analysis revealed that the postoperative survival of the LMTK3-negative group was superior to that of the LMTK3-positive group (P=0.043). Moreover, the multivariate analysis identified LMTK3 expression as an independent prognostic factor for patients with gastric cancer (P=0.019). These findings suggested that the expression of LMTK3 may be a negative prognostic factor in patients with gastric cancer. Moreover, targeting LMTK3 is a potential strategy for the treatment of gastric cancer, although the biological functions of LMTK3 in gastric cancer require further investigation.

Serum B7-H4 expression is a significant prognostic indicator for patients with gastric cancer.

BACKGROUND: B7-H4 is a novel B7 ligand that plays an important role in the T cell-mediated immune response as a negative regulator. Previous studies have suggested the aberrant expression of membrane B7-H4 in tumor cells. The aim of this study is to determine the expression levels of preoperative soluble B7-H4 (sB7-H4) in circulation and to investigate the correlations between sB7-H4 levels and clinicopathological parameters as well as the survival rate of patients with gastric cancer. METHODS: Blood specimens from 132 patients with gastric cancer and 63 healthy volunteers were analyzed by sandwich enzyme-linked immunosorbent assay. RESULTS: Median concentrations of sB7-H4 in patients with gastric cancer were significantly higher than those in healthy volunteers (16.85 versus 10.46 ng/mL; P = 0.008). Median levels of sB7-H4 were significantly correlated with tumor size, lymph node metastasis, the depth of tumor invasion and tumor-node-metastasis classification (P = 0.002, P = 0.001, P = 0.041 and P <0.001, respectively), but not with sex, age, tumor location or histological subtype (all P >0.05). Additionally, the overall survival rate was significantly lower in patients with high sB7-H4 levels when compared with low sB7-H4 levels (50.0% versus 77.3%, χ2 = 10.78, P = 0.001). Moreover, multivariate analysis demonstrated that the risk of death was significantly higher in patients with high sB7-H4 levels than in those with low sB7-H4 levels (P = 0.039). CONCLUSIONS: sB7-H4 is a valuable blood marker for predicting the progression and prognosis of patients with gastric cancer.

Lemur tyrosine kinase-3 is a significant prognostic marker for patients with colorectal cancer.

Lemur tyrosine kinase-3 (LMTK3) belongs to the family of serine-threonine-tyrosine kinases and the aberrant expression of LMTK3 was observed in several human malignancies. However, the association of LMTK3 with clinical outcomes in colorectal cancer patients is unclear. Thus, this present study was to evaluate the association of LMTK3 expression level with clinicopathologic factors and prognosis of patients with colorectal cancer (CRC). The expression level of LMTK3 in 69 archival paraffin-embedded colorectal tumor tissue specimens was examined by immunohistochemistry (IHC). As a result, we found that the LMTK3 expression level was significantly elevated in CRC tissues as compared with Crohn's disease or colorectal polyp tissues (P<0.0001, P<0.0001, respectively). Positive LMTK3 signals in the colorectal cancer cells were observed in about 89.9% (62 of 69) CRC tissue specimens. Additionally, LMTK3 expression was significantly correlated with lymph node metastasis and tumor-node-metastasis (TNM) classification (P=0.003, and P=0.008, respectively), but not with sex, age, tumor location, histological differentiation, tumor size, or depth of tumor invasion (all P>0.05). Kaplan-Meier survival curves showed that the overall survival rate was significantly higher in the patients with low expression of LMTK3 when compared with those patients with high LMTK3 (P=0.010). Moreover, multivariate analysis revealed that LMTK3 expression was an independent prognostic factor for CRC patients (P=0.047). These results suggest that LMTK3 protein could serve as a prognostic marker for CRC patients.

Serum lemur tyrosine kinase 3 expression in colorectal cancer patients predicts cancer progression and prognosis.

Lemur tyrosine kinase-3 (LMTK3) is a member of the families of serine-threonine-tyrosine kinases, which has been suggested to be a possible target and marker of breast cancer. However, its definitive physiopathological function in colorectal cancer (CRC) is poorly understood at present. The aim of this study was to determine the expression levels of preoperative-soluble LMTK3 (sLMTK3) in patients' blood with CRC and to subsequently evaluate whether or not its level in serum can be used to predict cancer progression and prognosis. The expression levels of sLMTK3 were measured by sandwich enzyme-linked immunosorbent assay in blood specimens from 60 patients with CRC and 53 healthy volunteers. As a result, we found that the mean concentration of sKMTK3 in CRC patients was significantly higher than that in healthy volunteers (P = 0.012). The expression levels of sLMTK3 were significantly correlated with histological subtype, depth of tumor invasion, and tumor-node-metastasis (TNM) classification (P = 0.038, 0.021, and 0.049, respectively), but not with sex, age, tumor location, tumor size, or lymph node metastasis. Additionally, Kaplan-Meier survival curves showed that patients with high levels of sLMTK3 had a poorer overall survival rate when compared with those of patients with low levels of sLMTK3 (P = 0.041). Moreover, multivariate analysis demonstrated that sLMTK3 expression and TNM stage were independent prognostic factors for CRC patients (P = 0.047 and 0.008, respectively). These results suggest that serum LMTK3 could be a valuable biomarker for predicting the progression and prognosis of patients with CRC.

Deguelin, a natural rotenoid, inhibits mouse myeloma cell growth in vitro via induction of apoptosis.

Deguelin is a naturally occurring rotenoid with strong cancer chemopreventive and antitumor activities. In the present study, we investigated the antitumor activity of deguelin against MPC-11 murine myeloma cells and the possible mechanism of action in vitro. Our results revealed that deguelin inhibited the proliferation of MPC-11 cells in a concentration- and time-dependent manner and caused the apoptotic death of MPC-11 cells. Following exposure to deguelin, the phosphorylation of Akt was decreased. The inhibition of cell growth may be associated with decreased levels of phosphorylated Akt. Deguelin-induced apoptosis was characterized by the upregulation of Bax, downregulation of Bcl-2 and activation of caspase-3. In conclusion, deguelin inhibits murine myeloma cell proliferation by inducing apoptosis via regulation of the Bax/Bcl-2 ratio and by inhibition of the activation of Akt. Its potential as an anticancer agent against multiple myeloma warrants further investigation.

Camptothecin nanocolloids based on N,N,N-trimethyl chitosan: efficient suppression of growth of multiple myeloma in a murine model.

Camptothecin (CPT) exhibits very strong antitumor effects by inhibiting the activity of DNA topoisomerase I, but its application is greatly limited due to its low solubility and the instability of the active lactone form. To overcome these shortcomings, in the present study, we prepared novel camptothecin nanocolloids based on N,N,N-trimethyl chitosan (CPT-TMC) to efficiently and safely administer CPT systemically. Herein, we investigated the antitumor activity of CPT-TMC against a murine Balb/c myeloma model. Our results showed that CPT-TMC more effectively inhibited tumor growth and prolonged survival time than CPT in vivo, but no statistical difference was observed in vitro between CPT-TMC and CPT. These findings suggest that N,N,N-trimethyl chitosan could increase the stability and the antitumor effect of CPT and CPT-TMC is a potential approach for the effective treatment of multiple myeloma.

Antitumor and antimetastatic activities of chloroquine diphosphate in a murine model of breast cancer.

Metastatic breast cancers are hard to treat and almost always fatal. Chloroquine diphosphate, a derivative of quinine, has long been used as a potent and commonly used medicine against different human diseases. We therefore investigated the effects of chloroquine diphosphate on a highly metastatic mouse mammary carcinoma cell line. In vitro treatment of 4T1 mouse breast cancer cells with chloroquine diphosphate resulted in significant inhibition of cellular proliferation and viability, and induction of apoptosis in 4T1 cells in a time- and dose-dependent manner. Further analysis indicated that induction of apoptosis was associated with the loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase. The effect of chloroquine diphosphate was then examined using a mice model in which 4T1 cells were implanted subcutaneously. Chloroquine diphosphate (25mg/kg and 50mg/kg, respectively) significantly inhibited the growth of the implanted 4T1 tumor cells and induced apoptosis in the tumor microenvironment. Moreover, the metastasis of tumor cells to the lungs was inhibited significantly and the survival of the mice enhanced. These data suggested that chloroquine diphosphate might have chemotherapeutic efficacy against breast cancer including inhibition of metastasis.

Barbigerone, a natural isoflavone, induces apoptosis in murine lung-cancer cells via the mitochondrial apoptotic pathway.

Barbigerone is a naturally occurring isoflavone with antioxidant activity. In present study, we investigated the antitumor activity of barbigerone against murine lung cancer cells LL/2 and the possible mechanism in vitro. Our results showed that barbigerone inhibited LL/2 cells proliferation in a concentration- and time-dependent manner and caused apoptotic death of LL/2 cells. Barbigerone-induced apoptosis was characterized by enhanced mitochondrial cytochrome c release, activation of caspase-3,-9, but not caspase-8. Exposure of LL/2 cells to barbigerone resulted in upregulation of Bcl-2-associated protein (Bax) and down-regulation of Bcl-2. In addition, proliferation inhibitory effect of barbigerone was associated with decreased level of phosphorylated p42/44 mitogen-activated protein kinase (p42/44 MAPK) and phosphorylated Akt. Moreover, barbigerone exhibit less toxicity to non-cancer cells than tumor cells. In conclusion, our results indicated that barbigerone can inhibit murine lung cancer cell proliferation by inducing apoptosis via mitochondrial apoptotic pathway and by decreasing phosphorylated p42/44 MAPK and Akt. Its potential to be a candidate of anti-cancer agent is worth being further investigated.

Chloroquine inhibits colon cancer cell growth in vitro and tumor growth in vivo via induction of apoptosis.

The present study was to investigate the anticancer effect of chloroquine on proliferation of mouse colon cancer cell line CT26 in vivo and in vitro and the possible mechanism. We found that chloroquine inhibited CT26 proliferation by concentration- and time-dependent manner. This effect was associated with apoptosis induction and decreased level of phosphorylated p42/44 mitogen-activated protein kinase and phosphorylated Akt. The in vivo study showed chloroquine-reduced tumor volume and prolonged survival time in CT26-bearing mice. These observations indicated chloroquine could inhibit CT26 proliferation by inducing apoptosis both in vitro and in vivo, providing its chemotherapeutic potential of human cancers.

Cell growth inhibition, G2/M cell cycle arrest, and apoptosis induced by chloroquine in human breast cancer cell line Bcap-37.

Chloroquine is an antimalarial drug that has been used in the treatment and prophylaxis of malaria since the 1950s. The present study was undertaken to examine the effects of chloroquine on Bcap-37 human breast cancer cells' growth, cell cycle modulation, apoptosis induction, and associated molecular alterations in vitro. The chloroquine treatment decreased the viability of Bcap-37 cells in a concentration- and time-dependent manner, which correlated with G(2)/M phase cell cycle arrest. The chloroquine-mediated cell cycle arrest was associated with a decrease in protein levels/activity of polo-like kinase 1 (Plk1), phosphorylated cell division cycle 25C (Cdc25C), phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated Akt. The chloroquine-treated Bcap-37 cells exhibited a marked decrease in the level of mitochondrial transmembrane potential (DeltaPsim), which was accompanied by the activation of caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). Exposure of Bcap-37 cells to chloroquine also resulted in the induction of spindle abnormalities. In conclusion, the findings in this study suggested that chloroquine might have potential anticancer efficacy, which could be attributed, in part, to its proliferation inhibition and apoptosis induction of cancer cells through modulation of apoptosis and cell cycle-related proteins expressions, down-regulation of mitochondrial transmembrane potential (DeltaPsim), and induction of spindle abnormalities.

[Effects of chloroquine diphosphate on proliferation and apoptosis of human leukemic K562 cells].

The purpose of this study was to investigate the effects of chloroquine diphosphate on the proliferation and apoptosis of human leukemic K562 cells, and to elucidate its possible mechanism of activity. The inhibitory effect of chloroquine diphosphate with different concentrations on K562 cell proliferation was detected by MTT method. Apoptosis was measured by flow cytometry (FCM); morphological analysis of apoptosis was performed after staining with propidium iodide (PI) under fluorescence microscope; cell apoptosis was assessed by the DNA ladder shown agarose gel electrophoresis. After treatment with chloroquine diphosphate, K562 cells were stained by Rhodamine 123 to detect changes in mitochondrial transmembrane potential (DeltaPsim) by FCM. The results showed that the cell viability decreased in dose-dependent manner, following chloroquine diphosphate treatment at different concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 micromol/L) for 24, 48 and 72 hours. By FCM analysis, the significant increases of sub-G(1) were observed. DNA ladder was detected and apoptotic nuclei were observed. DeltaPsim decreased in K562 cells after chloroquine diphosphate treatment. It is concluded that the chloroquine diphosphate can inhibit the proliferation of K562 cells and induce cell apoptosis, which may relate to down-regulation of mitochondrial transmembrane potential (DeltaPsim).