Kang-Ai Injection Inhibits Gastric Cancer Cells Proliferation through IL-6/STAT3 Pathway.

OBJECTIVE: To explore the mechanisms underlying the proliferative inhibition of Chinese herbal medicine Kang-Ai injection (KAI) in gastric cancer cells. METHODS: Gastric cancer cell lines MGC803 and BGC823 were treated by 0, 0.3%, 1%, 3% and 10% KAI for 24, 48 and 72 h, respectively. The cell proliferation was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The apoptosis and cell cycle were evaluated by flow cytometry. Interleukin (IL)-6 mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA), respectively. The protein expression levels of cyclin A, cyclin E, cyclin B1, cyclin D1, p21, retinoblastoma (RB), protein kinase B (AKT), extracellular regulated protein kinases (ERK), signal transducer and activator of transcription (STAT) 1 and STAT3 were detected by Western blot. RESULTS: KAI inhibited the proliferation of MGC803 and BGC823 gastric cancer cells in dose- and time-dependent manner. After treated with KAI for 48 h, the proportion of G1 phase was increased, expression level of cyclin D1 and phosphorylation-RB were down-regulated, whereas the expression of p21 was up-regulated (all P<0.01). Furthermore, 48-h treatment with KAI decreased the phosphorylation level of STAT3, inhibited the mRNA and protein expressions of IL-6 (all P<0.01). IL-6 at dose of 10 ng/mL significantly attenuated the proliferative effect of both 3% and 10% KAI, and recovered KAI-inhibited STAT3 phosphorylation and cyclin D1 expression level (all P<0.01). CONCLUSION: KAI exerted an anti-proliferative function by inhibiting IL-6/STAT3 signaling pathway followed by the induction of G1 phase arrest in gastric cancer cells.

FEN1 knockdown improves trastuzumab sensitivity in human epidermal growth factor 2-positive breast cancer cells.

Trastuzumab has been widely applied as a treatment for human epidermal growth factor 2 (HER2)-overexpressing breast cancer. However, the therapeutic efficacy of trastuzumab is limited. Flap endonuclease 1 (FEN1) is a multifunctional endonuclease that has a crucial role in DNA recombination and repair. Inhibition of FEN1 is associated with the reversal of anticancer drug resistance. However, it is unclear whether FEN1 is involved in trastuzumab resistance. In the present study, it was demonstrated that trastuzumab increases the expression of FEN1, and FEN1 knockdown significantly enhanced the sensitivity of BT474 cells to trastuzumab (P<0.05). It was also revealed that trastuzumab induced HER receptor activation, increased binding with FEN1 and estrogen receptor α (ERα), and upregulated ERα-target gene transcription (P<0.05). Upon silencing of FEN1 expression with siRNA, activation of HER receptor and FEN1 binding to ERα were decreased, and trastuzumab-induced ERα target gene upregulation was partially ameliorated (P<0.05). These results suggest that FEN1 may mediate trastuzumab resistance via inducing HER receptor activation and enhancing ERα-target gene transcription. The findings of the present study indicate a novel role of FEN1 in trastuzumab resistance, suggesting that targeting FEN1 may enhance the efficiency of trastuzumab as a treatment for HER2-positive breast cancer.

2-Aminophenoxazine-3-one-induced apoptosis via generation of reactive oxygen species followed by c-jun N-terminal kinase activation in the human glioblastoma cell line LN229.

2-Aminophenoxazine-3-one (Phx-3) induces apoptosis in several types of cancer cell lines. However, the mechanism of apoptosis induction by Phx-3 has not been fully elucidated. In this study, we investigated the anticancer effects of Phx-3 in the glioblastoma cell line LN229 and analyzed its molecular mechanism. The results indicated that 6- and 20-h treatment with Phx-3 significantly induced apoptosis in LN229 cells, with downregulation of survivin and XIAP. Both ERK and JNK, which are the members of the MAPK family, were activated after treatment with Phx-3. Inhibition of ERK using the specific inhibitor U0126 blocked the Phx-3-induced apoptosis only in part. However, inhibition of JNK using the specific inhibitor SP600125 completely prevented Phx-3-induced apoptosis and restored the phosphorylation states of ERK to the control levels. Enhanced generation of reactive oxygen species (ROS) was detected after 3-h treatment with Phx-3. In addition, the ROS scavenger melatonin almost completely blocked Phx-3-induced JNK activation and apoptosis. This suggests that JNK activation was mediated by Phx-3-induced ROS generation. Although SP600125 and melatonin completely blocked the reduction of mitochondrial membrane potential after a 3-h treatment with Phx-3, extension of Phx-3 exposure time to 20 h resulted in no cancelation of mitochondrial depolarization by these reagents. These reagents also had little effect on the decreased expression of survivin and XIAP during a 3-20-h exposure to Phx-3. These results indicate that the production of ROS following JNK activation is the main axis of Phx-3-induced apoptosis in LN229 cells for short-term exposure to Phx-3, whereas alternative mechanism(s) appear to be involved in apoptosis induction during long-term exposure to Phx-3.

VEGF expression is augmented by hypoxia­induced PGIS in human fibroblasts.

Prostacyclin synthase (PGIS or PTGIS) is an enzyme that catalyses the conversion of prostaglandin H2 (PGH2) to prostaglandin I2 (PGI2). PGI2 promotes cancer growth by activating peroxisome proliferator-activated receptor δ (PPARδ), and increases the expression levels of the pro-angiogenic factor vascular endothelial growth factor (VEGF). We found that the expression of the PGIS gene was enhanced in WI-38, TIG-3-20 and HEL human lung fibroblast cells and two cancer cell lines (NB-1 and G361) under hypoxic conditions. The main localization of PGIS changed from the cytoplasm to the nucleus by hypoxia in WI-38 cells. The induced PGIS had an enzymatic activity since the intracellular level of 6-keto-prostaglandin, a useful marker of PGI2 biosynthesis in vivo, was increased with the increasing levels of PGIS. Expression of VEGF was increased in parallel with PGIS induction under hypoxic conditions. PGIS knockdown resulted in the decreased expression of VEGF mRNA. Since VEGF is a known PPARδ target gene, we examined the effects of siRNAs targeting PPARδ on the expression of VEGF under hypoxic conditions. Knockdown of PPARδ suppressed the expression of VEGF under hypoxic conditions in WI-38 cells. These findings suggest that PGIS is induced by hypoxia and regulates the expression of VEGF in fibroblasts. Fibroblasts in the hypoxic area of tumors may have an important role in tumor growth and angiogenesis.

Crucial involvement of tumor-associated neutrophils in the regulation of chronic colitis-associated carcinogenesis in mice.

Ulcerative colitis (UC) is a major form of chronic inflammation that can frequently progress to colon cancer. Several studies have demonstrated massive infiltration of neutrophils and macrophages into the lamina propria and submucosa in the progression of UC-associated colon carcinogenesis. Macrophages contribute to the development of colitis-associated colon cancer (CAC). However, the role of neutrophils is not well understood. To better understand the involvement of tumor-associated neutrophils (TANs) in the regulation of CAC, we used a mouse CAC model produced by administering azoxymethane (AOM), followed by repeated dextran sulfate sodium (DSS) ingestion. This causes severe colonic inflammation and subsequent development of multiple tumors in mice colon. We observed that colorectal mucosal inflammation became increasingly severe with AOM and DSS treatment. Macrophages infiltrated the lamina propria and submucosa, together with a marked increase in neutrophil infiltration. The chemokine CXCL2 increased in the lamina propria and submucosal regions of the colons of the treated mice, together with the infiltration of neutrophils expressing CXCR2, a specific receptor for CXCL2. This process was followed by neoplastic transformation. After AOM and DSS treatment, the mice showed enhanced production of metalloproteinase (MMP)-9 and neutrophil elastase (NE), accompanied by excessive vessel generation and cell proliferation. Moreover, CXCL2 promoted neutrophil recruitment and induced neutrophils to express MMP-9 and NE in vitro. Furthermore, administration of neutrophil-neutralizing antibodies after the last DSS cycle markedly reduced the number and size of tumors and decreased the expression of CXCR2, CXCL2, MMP-9, and NE. These observations indicate a crucial role for TANs in the initiation and progression of CAC and suggest that the CXCL2-CXCR2 axis might be useful in reducing the risk of UC-associated colon cancer.

The association between common genetic variant of microRNA-146a and cancer susceptibility.

Published data on the association between microRNA-146a (miR-146a) G/C polymorphism and cancer susceptibility are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. A total of 23 studies including 10,585 cases and 12,183 controls were used in the meta-analysis. Overall, no significant associations were found between miR-146a G/C polymorphism and cancer risk when all studies pooled into the meta-analysis (GC vs. CC: OR=1.08, 95% CI=0.94-1.24; GG vs. CC: OR=1.13, 95% CI=0.93-1.37; dominant model: OR=1.09, 95% CI=0.94-1.26). In the subgroup analysis by ethnicity, still no significant associations were found. In the subgroup analysis by cancer type, statistically significantly increased risks were found for papillary thyroid carcinoma (GC vs. CC: OR=3.44, 95% CI=1.86-6.34; GG vs. CC: OR=2.20, 95% CI=1.22-3.99; dominant model: OR=2.68, 95% CI=1.48-4.83). In the subgroup analysis by population-based controls or hospital-based controls, no statistically significantly increased risks were found. Despite some limitations, this meta-analysis suggests that the miR-146a G allele is a low-penetrant risk factor for papillary thyroid carcinoma development.

2-Aminophenoxazine-3-one and 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one cause cellular apoptosis by reducing higher intracellular pH in cancer cells.

We examined intracellular pH (pHi) of ten cancer cell lines derived from different organs and two normal cell lines including human embryonic lung fibroblast cells (HEL) and human umbilical vein endothelial cells (HUVEC) in vitro, and found that pHi of most of these cancer cells was evidently higher (pH 7.5 to 7.7) than that of normal cells (7.32 and 7.44 for HEL and HUVEC, respectively) and that of primary leukemic cells and erythrocytes hitherto reported (≤7.2). Higher pHi in these cancer cells could be related to the Warburg effect in cancer cells with enhanced glycolytic metabolism. Since reversal of the Warburg effect may perturb intracellular homeostasis in cancer cells, we looked for compounds that cause extensive reduction of pHi, a major regulator of the glycolytic pathway and its associated metabolic pathway. We found that phenoxazine compounds, 2-aminophenoxazine-3-one (Phx-3) and 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one (Phx-1) caused a rapid and drastic dose-dependent decrease of pHi in ten different cancer cells within 30 min, though the extent of the decrease of pHi was significantly larger for Phx-3 (ΔpHi = 0.6 pH units or more for 100 µM Phx-3) than for Phx-1 (ΔpHi = 0.1 pH units or more for 100 µM Phx-1). This rapid and drastic decrease of pHi in a variety of cancer cells caused by Phx-3 and Phx-1 possibly perturbed their intracellular homeostasis, and extensively affected the subsequent cell death, because these phenoxazines exerted dose-dependent proapoptotic and cytotoxic effects on these cells during 72 h incubation, confirming a causal relationship between ΔpHi and cytotoxic effects due to Phx-3 and Phx-1. Phx-3 and Phx-1 also reduced pHi of normal cells including HEL and HUVEC, although they exerted less proapoptotic and cytotoxic effects on these cells than on cancer cells. Drugs such as Phx-3 and Phx-1 that reduce pHi and thereby induce cellular apoptosis might serve as benevolent anticancer drugs.

GSTM1 null allele is a risk factor for gastric cancer development in Asians.

Glutathione S-transferase M1 (GSTM1), which plays an important role in detoxification pathways to protect against damage caused by reactive metabolites of chemicals, has been considered as potential gastric cancer susceptibility genes. However, the published data on the association between GSTM1 present/null polymorphism and gastric cancer risk are still inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. Totally, 44 studies including 5440 cases and 11607 controls were involved in the analysis. When all studies were pooled into the meta-analysis, obviously increased gastric cancer risk was found in null genotype carriers (OR=1.19, 95% CI: 1.08-1.33). When stratified by ethnicity, obviously evaluated risk was found in Asians (OR=1.31, 95% CI: 1.11-1.54) but not reached to statistically significance in Caucasians (OR=1.11, 95% CI: 0.96-1.28). In the subgroup analysis by hospital-based studies or population-based studies, statistically significantly elevated risk was found in hospital-based studies (OR=1.34, 95% CI: 1.07-1.67) but not reached to statistically significance in population-based studies (OR=1.11, 95% CI: 0.99-1.25). In summary, this meta-analysis result indicates that the GSTM1 null genotype is a low-penetrant risk factor for gastric cancer development in Asians.

The p21 Ser31Arg polymorphism and breast cancer risk: a meta-analysis involving 51,236 subjects.

Published data on the association between p21 Ser31Arg polymorphism and breast cancer risk are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. Crude ORs with 95% CIs were used to assess the strength of association between the p21 Ser31Arg polymorphism and breast cancer risk. The pooled ORs were performed for co-dominant model (Ser/Arg vs. Ser/Ser, Arg/Arg vs. Ser/Ser), dominant model (Arg/Arg + Ser/Arg vs. Ser/Ser), and recessive model (Arg/Arg vs. Ser/Arg + Ser/Ser). A total of 21 studies including 22,109 cases and 29,127 controls were involved in this meta-analysis. Overall, no significant associations were found between p21 Ser31Arg polymorphism and breast cancer risk when all studies pooled into the meta-analysis. In the subgroup analysis by ethnicity, significantly increased risk was found for Caucasians (Arg/Arg vs. Ser/Ser: OR 1.496, 95% CI 1.164-1.924; and recessive model: OR 1.492, 95% CI 1.161-1.919). When stratified by study design, statistically significantly elevated risk was found for population-based studies (Ser/Arg vs. Ser/Ser: OR 1.085, 95% CI 1.019-1.156). In conclusion, this meta-analysis suggests that the p21 Ser31Arg polymorphism may be associated with breast cancer development in Caucasian. However, large sample and representative population-based studies with homogeneous breast cancer patients and well-matched controls are warranted to confirm this finding.

2-Aminophenoxazine-3-one induces cellular apoptosis by causing rapid intracellular acidification and generating reactive oxygen species in human lung adenocarcinoma cells.

2-Aminophenoxazine-3-one (Phx-3)-induced apoptosis was investigated. Phx-3 suppressed the viability of human lung adenocarcinoma cell line A549 and induced cellular apoptosis 6 h after treatment. Prior to these events, intracellular pH (pHi) was rapidly decreased from pH 7.65 to 7.10 within 30 min when A549 cells were treated with 7 microM Phx-3. This intracellular acidification continued for 3 h in the cells. Augmented production of reactive oxygen species (ROS) was obseved 1 h after treatment of A549 cells with 7 microM Phx-3, and cell cycle arrest at G1 was indicated 3 h after treatment. The translocation of NF-kappaB from the cytosol to the nucleus was clearly indicated 1 h after the administration of Phx-3 to A549 cells, while it was significantly suppressed when Nac, a scavenger of ROS, was added to the cells with Phx-3. The Phx-3-induced apoptosis in A549 cells was significantly suppressed when Nac was administered to the cells. These results suggest that a decrease of pHi, caused by depolarization of the mitochondria, may trigger the dysfunction of mitochondria causing ROS production; therefore, both the translocation of NF-kappaB from the cytoplasm to the nucleus and apoptosis induction were promoted in A549 cells. Microscopic examination of the cellular localization of Phx-3 in A549 cells revealed that Phx-3 was mainly localized in the cytoplasm and the mitochondria, but not in the nucleus. The present results indicate that Phx-3 might be a strong anticancer drug against lung cancer, which is intractable to chemotherapy, by causing various early events, including the decrease of pHi and ROS production, and finally inducing cellular apoptosis.

Overexpression of survivin in primary ATL cells and sodium arsenite induces apoptosis by down-regulating survivin expression in ATL cell lines.

Patients with acute- or lymphoma-type adult T-cell leukemia (ATL) have a poor outcome because of the intrinsic drug resistance to chemotherapy. Protection from apoptosis is a common feature involved in multidrug-resistance of ATL. IAP (inhibitor of apoptosis) family proteins inhibit apoptosis induced by a variety of stimuli. In this study, we investigated the expression of IAP family members (survivin, cIAP1, cIAP2, and XIAP) in the primary leukemic cells from patients with ATL. We found that survivin was overexpressed in ATL, especially in acute-type ATL. Sodium arsenite was shown to down-regulate the expression of survivin at both the protein and RNA levels in a time- and dose-dependent manner, thus inhibiting cell growth, inducing apoptosis, and enhancing the caspase-3 activity in ATL cells. Nuclear factor-kappaB (NF-kappaB) enhances the transcriptional activity of survivin. Sodium arsenite suppressed the constitutive NF-kappaB activation by preventing the IkappaB-alpha degradation and the nuclear translocation of NF-kappaB. These findings suggest that survivin is an important antiapoptotic molecule that confers drug resistance on ATL cells. Sodium arsenite was shown to down-regulate the expression of survivin through the NF-kappaB pathway, thus inhibiting cell growth and promoting apoptosis of ATL cells.

Protection against DNA damage-induced apoptosis by the angiogenic factor thymidine phosphorylase.

Thymidine phosphorylase (TP) is involved both in pyrimidine nucleoside metabolism and in angiogenesis. TP also conferred the resistance to hypoxia-induced apoptosis of the cancer cells. In U937 cells, DNA damage-inducing agents significantly enhanced the expression of TP. Cell lines stably transfected with TP cDNA were more resistant to the DNA damage-inducing agents than the mock-transfected cells and showed augmented activity of Akt. The cytoprotective function of TP against DNA damage was independent of its enzymatic activity. The resistance to apoptosis was partially abrogated by treatment with the phosphatidyl inositol 3-kinase (PI3K) inhibitors, suggesting that the cytoprotective function of TP is mediated, at least in part, by regulation of the PI3K/Akt pathway. These findings indicate that TP expression in increased by various stress including DNA damage and that TP molecules confer resistance to DNA damage-induced apoptosis in cancer cells.

Cepharanthine potently enhances the sensitivity of anticancer agents in K562 cells.

A major impediment to cancer treatment is the development of resistance by the tumor. P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) are involved in multidrug resistance. In addition to the extrusion of chemotherapeutic agents through these transporters, it has been reported that there are differences in the intracellular distribution of chemotherapeutic agents between drug resistant cells and sensitive cells. Cepharanthine is a plant alkaloid that effectively reverses resistance to anticancer agents. It has been previously shown that cepharanthine is an effective agent for the reversal of resistance in P-gp-overexpressing cells. Cepharanthine has also been reported to have numerous pharmacological effects besides the inhibition of P-gp. It has also been found that cepharanthine enhanced sensitivity to doxorubicin (ADM) and vincristine (VCR), and enhanced apoptosis induced by ADM and VCR of P-gp negative K562 cells. Cepharanthine changed the distribution of ADM from cytoplasmic vesicles to nucleoplasm in K562 cells by inhibiting the acidification of cytoplasmic organelles. Cepharanthine in combination with ADM should be useful for treating patients with tumors.

Thymidine phosphorylase suppresses apoptosis induced by microtubule-interfering agents.

We investigated the ability of thymidine phosphorylase (TP) to confer cancer cells resistance to MIA (microtubule-interfering agents)-induced apoptosis. Jurkat cells were stably transfected with TP cDNA (Jurkat/TP) and the sensitivity to MIAs were examined. Jurkat/TP cells were more resistant to apoptosis induced by nocodazole, vincristine, vinblastine, paclitaxel and 2-methoxyestradiol than mock-transfected Jurkat/CV cells. TP enzymatic activity was not required for this effect of TP. Jurkat/TP cells showed weak phosphorylation of Bcl-2, and kinase inhibitors staurosporine and genistein attenuated not only MIA-induced Bcl-2 phosphorylation but also cytotoxicity of MIA in Jurkat/CV, but not in Jurkat/TP. MIAs diminished expression of FasL in Jurkat/TP but not in Jurkat/CV, and neutralization of FasL by anti-FasL antibody considerably attenuated the cytotoxic effect of the MIAs in Jurkat/CV, but the effect of the antibody was marginal in Jurkat/TP cells. Our study provides further evidence that TP functions in conferring resistance on cancer cells to the stress induced by MIAs. In addition, we show that TP-induced inhibition of Bcl-2 phosphorylation and suppression of FasL may contribute to the protective function of TP in cancer cells.

Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes.

Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs. To date, little is known about the process of vault assembly or about the stability of vault components. In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells. Using an immunoprecipitation assay, we found that it took more than 4h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5h. Once incorporated into the vault complex, both MVP and VPARP were very stable. Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology. The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles. This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles. In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly.

Reversal of P-glycoprotein mediated multidrug resistance by a newly synthesized 1,4-benzothiazipine derivative, JTV-519.

A newly synthesized 1,4-benzothiazipine derivate, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2,3,4,5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) mediated multidrug resistance (MDR) in K562/MDR and KB/MRP cells, respectively. JTV-519 at 3 microM reversed the resistance of K562/MDR cells to vincristine (VCR), taxol, etoposide (VP16), adriamycin (ADM) and actinomycin D and at 0.5 or 1 microM reversed their resistance to STI571. JTV-519 at 10 microM enhanced the accumulation of ADM in K562/MDR cells to the level in parental K562 cells and inhibited the efflux of ADM from K562/MDR cells. Photoaffinity labeling of P-gp with 3H-azidopine was almost completely inhibited by 500 microM JTV-519. JTV-519 at 3 microM also partially reversed the resistance of KB/MRP cells to VCR and at 500 microM partially inhibited the photoaffinity labeling of MRP1 with (125)I-II-azidophenyl agosterol A (125I-azidoAG-A). These results suggest that JTV-519 reversed the resistance to the anti-cancer agents in P-gp and MRP1 overexpressing multidrug-resistant cells by directly binding to P-gp and MRP1, and competitively inhibiting transport of the anti-cancer agents.