TOB1 inhibits the gastric cancer progression by focal adhesion pathway and ERK pathway based on transcriptional and metabolic sequencing.

Gastric cancer is one of the most malignant digestive tract tumors worldwide and its progression is associated with gene expression and metabolic alteration. We revealed that the gastric cancer patients with lower expression level of TOB1 exhibited poorer overall survivals according to the data in Kaplan-Meier Plotter. The unphosphorylated TOB1 protein which is effective expressed lower in gastric cancer cells. The gastric cancer cells with TOB1 gene depletion performed higher abilities of proliferation, migration and invasion and lower ability of apoptosis in vitro. The TOB1 gene depletion also promoted the tumorigenesis of gastric cancer cells in vivo. The gastric cancer cells with TOB1 gene overexpression had the converse behaviors. The transcriptional and metabolic sequencing was performed. The analyzation results showed that genes correlate-expressed with TOB1 gene were enriched in the pathways related to ERK pathway, including focal adhesion pathway, which was verified using real-time quantitative PCR. After inhibiting ERK pathway, the proliferation, colony formation and migration abilities were reduced in gastric cancer cells with low phosphorylated TOB1 protein expression level. Moreover, Pearson correlation analysis was adopted to further analyze the correlation of enriched metabolic products and differentially expressed genes. The expression of Choline, UDP-N-acetylglucosamine, Adenosine and GMP were related to the function of TOB1. This study demonstrates the genes and metabolites related to focal adhesion pathway and ERK pathway are the potential diagnosis and therapeutic targets to gastric cancer with TOB1 depletion.

Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Novel insights into the ecDNA formation mechanism involving MSH3 in methotrexate­resistant human colorectal cancer cells.

Extrachromosomal DNAs (ecDNAs), also known as double minutes (DMs), can induce a fast increase in gene copy numbers and promote the development of cancer, including drug resistance. MutS homolog 3 (MSH3), a key protein in mismatch repair, has been indicated to participate in the regulation of DNA double­strand break (DSB) repair, which has been reported to be associated with the formation of ecDNAs. However, it remains unclear whether MSH3 can influence drug resistance via ecDNAs in cancer. In the present study, high MSH3 expression was observed in methotrexate (MTX)­resistant HT29 cells [DM­ and homogeneously staining region (HSR)­containing cells] compared with parental HT29 cells. Additionally, decreased amounts of ecDNAs, HSRs and amplified genes locating on ecDNAs and HSRs were detected following depletion of MSH3 and this could be reversed by overexpressing MSH3 in DM­containing cells. No corresponding changes were found in HSR­containing cells. The present study further verified the involvement of MSH3­regulated DNA DSB repair pathways in the formation of ecDNAs by detecting the expression of core proteins and pathway activity. Furthermore, expulsion of ecDNAs/HSRs was detected and increased frequencies of micronuclei/nuclear buds with dihydrofolate reductase (DHFR) signals were observed in MSH3­depleted DM­containing cells. Finally, changes in MSH3 expression could affect DHFR amplification­derived DHFR expression and cell sensitivity to MTX, suggesting that MSH3 may influence cancer drug resistance by altering the amount of ecDNAs. In conclusion, the present study revealed a novel mechanism involving MSH3 in the regulation of ecDNAs by DSB repair, which will have clinical value in the treatment of ecDNA­based drug resistance in cancer.

Contribution of PGAP3 co-amplified and co-overexpressed with ERBB2 at 17q12 involved poor prognosis in gastric cancer.

The locus at 17q12 erb-b2 receptor tyrosine kinase 2 (ERBB2) has been heavily amplificated and overexpressed in gastric cancer (GC), but it remains to be elucidated about the clinical significance of the co-amplification and co-overexpression of PGAP3 gene located around ERBB2 in GC. The profile of PGAP3 and ERBB2 in four GC cell lines and tissue microarrays containing 418 primary GC tissues was assessed to investigate the co-overexpression and clinical significance of the co-amplified genes, and to evaluate the impact of the co-amplified genes on the malignancy of GC. Co-amplification of PGAP3 and ERBB2 accompanied with co-overexpression was observed in a haploid chromosome 17 of NCI-N87 cells with double minutes (DMs). PGAP3 and ERBB2 were overexpressed and positively correlated in 418 GC patients. Co-overexpression of the PGAP3 and ERBB2 was correlated with T stage, TNM stage, tumour size, intestinal histological type and poor survival proportion in 141 GC patients. In vitro, knockdown of the endogenous PGAP3 or ERBB2 decreased cell proliferation and invasion, increased G1 phase accumulation and induced apoptosis in NCI-N87 cells. Furthermore, combined silencing of PGAP3 and ERBB2 showed an additive effect on resisting proliferation of NCI-N87 cells compared with targeting ERBB2 or PGAP3 alone. Taken together, the co-overexpression of PGAP3 and ERBB2 may be crucial due to its significant correlation with clinicopathological factors of GC. Haploid gain of PGAP3 co-amplified with ERBB2 is sufficient to facilitate the malignancy and progression of GC cells in a synergistic way.

Inhibiting homologous recombination decreases extrachromosomal amplification but has no effect on intrachromosomal amplification in methotrexate-resistant colon cancer cells.

Gene amplification, which involves the two major topographical structures double minutes (DMs) and homegeneously stained region (HSR), is a common mechanism of treatment resistance in cancer and is initiated by DNA double-strand breaks. NHEJ, one of DSB repair pathways, is involved in gene amplification as we demonstrated previously. However, the involvement of homologous recombination, another DSB repair pathway, in gene amplification remains to be explored. To better understand the association between HR and gene amplification, we detected HR activity in DM- and HSR-containing MTX-resistant HT-29 colon cancer cells. In DM-containing MTX-resistant cells, we found increased homologous recombination activity compared with that in MTX-sensitive cells. Therefore, we suppressed HR activity by silencing BRCA1, the key player in the HR pathway. The attenuation of HR activity decreased the numbers of DMs and DM-form amplified gene copies and increased the exclusion of micronuclei and nuclear buds that contained DM-form amplification; these changes were accompanied by cell cycle acceleration and increased MTX sensitivity. In contrast, BRCA1 silencing did not influence the number of amplified genes and MTX sensitivity in HSR-containing MTX-resistant cells. In conclusion, our results suggest that the HR pathway plays different roles in extrachromosomal and intrachromosomal gene amplification and may be a new target to improve chemotherapeutic outcome by decreasing extrachromosomal amplification in cancer.

Combined caveolin-1 and epidermal growth factor receptor expression as a prognostic marker for breast cancer.

Previous studies have indicated that caveolin-1 (Cav-1) is able to bind the signal transduction factor epidermal growth factor receptor (EGFR) to regulate its tyrosine kinase activity. The aim of the present study was to evaluate the clinical significance of Cav-1 gene expression in association with the expression of EGFR in patients with breast cancer. Primary breast cancer samples from 306 patients were analyzed for Cav-1 and EGFR expression using immunohistochemistry, and clinical significance was assessed using multivariate Cox regression analysis, Kaplan-Meier estimator curves and the log-rank test. Stromal Cav-1 was downregulated in 38.56% (118/306) of tumor tissues, whereas cytoplasmic EGFR and Cav-1 were overexpressed in 53.92% (165/306) and 44.12% (135/306) of breast cancer tissues, respectively. EGFR expression was positively associated with cytoplasmic Cav-1 and not associated with stromal Cav-1 expression in breast cancer samples; however, low expression of stromal Cav-1 was negatively associated with cytoplasmic Cav-1 expression in total tumor tissues, and analogous results were identified in the chemotherapy group. Multivariate Cox's proportional hazards model analysis revealed that, for patients in the estrogen receptor (ER)(+) group, the expression of stromal Cav-1 alone was a significant prognostic marker of breast cancer. However, in the chemotherapy, human epidermal growth factor receptor 2 (HER-2)(-), HER-2(+) and ER(-) groups, the use of combined markers was more effective prognostic marker. Stromal Cav-1 has a tumor suppressor function, and the combined marker stromal Cav-1/EGFR expression was identified as an improved prognostic marker in the diagnosis of breast cancer. Parenchymal expression of Cav-1 is able to promote EGFR signaling in breast cancer, potentially being required for EGFR-mediated initiation of mitosis.

Decreased TOB1 expression and increased phosphorylation of nuclear TOB1 promotes gastric cancer.

TOB1, a member of the BTG/TOB protein family, inhibits tumor cell proliferation. We previously observed down-regulation and phosphorylation of TOB1 in gastric cancer (GC). Here, we examined the subcellular distribution and clinical significance of TOB1 expression and phosphorylation in GC. Immunohistochemical analysis of 341 primary GC and corresponding normal gastric tissue samples demonstrated that nuclear TOB1 expression was lower in GC than normal tissue (80.4% vs. 92.4%), and decreased nuclear TOB1 expression correlated with high TNM stage. By contrast, phosphorylation of nuclear TOB1 was higher in GC than normal gastric tissue (66.0% vs. 36.4%), and was associated with poorly differentiated and high TNM stage tumors. Patients with intestinal type GC and increased nuclear TOB1 phosphorylation had poor overall survival. Multivariate survival analysis indicated the nuclear concentration of phosphorylated TOB1 was an independent prognostic factor for intestinal type GC. Overexpression of TOB1 containing mutations in its nuclear export signal inhibited GC cell proliferation, migration, and invasion compared to cells expressing TOB1 with the nuclear localization signal. Thus, decreased TOB1 expression and increased phosphorylation of nuclear TOB1 is associated with aggressive tumor behavior and poor prognosis in intestinal type GC. Additionally, TOB1 nuclear retention is critical for its anti-proliferative activity.

TRIB1 promotes colorectal cancer cell migration and invasion through activation MMP-2 via FAK/Src and ERK pathways.

Colorectal cancer (CRC) is the third most common cancer in the world and distant metastasis is the leading cause of death among CRC patients. However, the underlying mechanisms of distant metastasis remain largely unknown. Amplification of 8q24 is a common chromosomal abnormality in CRC. In the present study, a putative oncogene at 8q24, TRIB1, was characterized for its role in CRC metastasis and underlying molecular mechanisms. Higher expression of TRIB1 protein was detected in 58/83 (69.9%) of CRC tissues, compared with adjacent non-tumor tissues. Moreover, the expression of TRIB1 was significantly associated with distant metastasis (P=0.043) and advanced staging (P=0.008) in CRC tissues. TRIB1 overexpression was also correlated with poor prognosis in CRC patients as analyzed in PrognoScan database. In addition, elevated expression of TRIB1 promoted CRC cell motility and adhesive ability, while silencing of TRIB1 reduced those effects. Further study revealed that TRIB1-mediated migration and invasion of CRC cells required up-regulation of MMP-2 through the activation of FAK/Src and ERK pathway. Collectively, the results suggest that TRIB1 promotes CRC cell motility by activation MMP-2 via the FAK/Src and ERK pathways. It may provide a potential diagnostic and therapeutic target for CRC.

SEI1 induces genomic instability by inhibiting DNA damage response in ovarian cancer.

Previous studies have shown that the oncogene SEI1 is highly expressed in ovarian carcinomas, and promoting genomic instability. However, the molecular mechanism of SEI1 in promoting genomic instability remains unclear. We observed SEI1 overexpression in 30 of 46 cases of ovarian cancer compared to non-tumor tissues and the overexpression of SEI1 was positively associated with the tumor FIGO stage. Our functional studies revealed that overexpression of SEI1 could induce genomic instability and increased DNA strand breaks. In contrast, SEI1 co-localized with γH2AX and phosphorylated ATM and DNAPKcs in the nucleus. Furthermore, we found that overexpression of SEI1 induced translocation of the SEI1 protein from the cytoplasm to the nucleus; ATM and DNAPKcs were associated with the cytoplasm-to-nucleus translocation of SEI1. To further prove the correlation between the DNA damage response (DDR) and SEI1, we knocked down SEI1 expression in SEI1-transfected ovarian cancer cell lines. The expression of DDR proteins was significantly downregulated, and the number of micronuclei was significantly decreased. Together, these results define a new mechanism of SEI1 in the regulation of genomic stability and in the malignant progression of ovarian cancer.

Met promotes the formation of double minute chromosomes induced by Sei-1 in NIH-3T3 murine fibroblasts.

BACKGROUND: Sei-1 is an oncogene capable of inducing double minute chromosomes (DMs) formation. DMs are hallmarks of amplification and contribute to oncogenesis. However, the mechanism of Sei-1 inducing DMs formation remains unelucidated. RESULTS: DMs formation significantly increased during serial passage in vivo and gradually decreased following culture in vitro. micro nuclei (MN) was found to be responsible for the reduction. Of the DMs-carrying genes, Met was found to be markedly amplified, overexpressed and highly correlated with DMs formation. Inhibition of Met signaling decreased the number of DMs and reduced the amplification of the DMs-carrying genes. We identified a 3.57Mb DMs representing the majority population, which consists of the 1.21 Mb AMP1 from locus 6qA2 and the 2.36 Mb AMP2 from locus 6qA2-3. MATERIALS AND METHODS: We employed NIH-3T3 cell line with Sei-1 overexpression to monitor and characterize DMs in vivo and in vitro. Array comparative genome hybridization (aCGH) and fluorescence in situ hybridization (FISH) were performed to reveal amplification regions and DMs-carrying genes. Metaphase spread was prepared to count the DMs. Western blot and Met inhibition rescue experiments were performed to examine for involvement of altered Met signaling in Sei-1 induced DMs. Genomic walking and PCR were adopted to reveal DMs structure. CONCLUSIONS: Met is an important promotor of DMs formation.

Expression of pY397 FAK promotes the development of non-small cell lung cancer.

Focal adhesion kinase (FAK) expression has been identified as associated with cancer development and metastasis. Autophosphorylation of FAK at tyrosine (Y) 397 (pY397) performs a critical role in tumor cell signaling. However, few studies have evaluated the expression of pY397 FAK in non-small cell lung cancer (NSCLC). In the present study, pY397 FAK expression in NSCLC was investigated using immunohistochemistry. pY397 FAK staining scores were compared between various groups of specimens and the associations between clinical and pathological characteristics were investigated. A Kaplan-Meier survival curve was used to determine the association between pY397 FAK expression and the prognosis of NSCLC patients. The results of the present study revealed that pY397 FAK expression was localized to the cytoplasm of lung cells, and that pY397 FAK was overexpressed in NSCLC tissues, as well as associated metastatic tissues, when compared with the corresponding non-tumor tissues. However, no significant difference was identified between the pY397 FAK expression in primary lesions and lymph node metastases. Furthermore, pY397 FAK staining scores were not found to be associated with the tumor size, gender, degree of differentiation, histotypes, presence of lymph node metastases or survival rate of NSCLC patients. These results indicate that pY397 FAK is involved with the development of NSCLC, but is not a prognostic marker for the disease.

Ribosomal L22-like1 (RPL22L1) Promotes Ovarian Cancer Metastasis by Inducing Epithelial-to-Mesenchymal Transition.

Double minute chromosomes (DMs) have important implications for cancer progression because oncogenes frequently amplified on them. We previously detected a functionally undefined gene amplified on DMs, Ribosomal L22-like1 (RPL22L1). The relationship between RPL22L1 and cancer progression is unknown. Here, RPL22L1 was characterized for its role in ovarian cancer (OC) metastasis and its underlying mechanism was examined. DNA copy number and mRNA expression of RPL22L1 in OC cells was analyzed using data obtained from The Cancer Genome Atlas and the Gene Expression Omnibus database. An immunohistochemical analysis of clinical OC specimens was performed and the relationships between expression level and clinicopathological factors were evaluated. Additionally, in vivo and in vitro assays were performed to understand the role of RPL22L1 in OC. RPL22L1 expression was higher in OC specimens than in normal tissues, and its expression level was highly positively correlated with invasion and lymph node metastasis (P < 0.05). RPL22L1 over-expression significantly enhanced intraperitoneal xenograft tumor development in nude mice and promoted invasion and migration in vitro. Additionally, RPL22L1 knockdown remarkably inhibited UACC-1598 cells invasion and migration. Further, RPL22L1 over-expression up-regulated the mesenchymal markers vimentin, fibronectin, and α-SMA, reduced expression of the epithelial markers E-cadherin, α-catenin, and ß-catenin. RPL22L1 inhibition reduced expression of vimentin and N-cadherin. These results suggest that RPL22L1 induces epithelial-to-mesenchymal transition (EMT). Our data showed that the DMs amplified gene RPL22L1 is critical in maintaining the aggressive phenotype of OC and in triggering cell metastasis by inducing EMT. It could be employed as a novel prognostic marker and/or effective therapeutic target for OC.

Association between sister chromatid exchange and double minute chromosomes in human tumor cells.

BACKGROUND: Double minute chromosomes (DMs) are the cytogenetic hallmark of extra-chromosomal genomic amplification. They can well represent the advanced stage of malignancy. However, the mechanisms of DM generation are still not fully understood. Here, the sister chromatid exchange (SCE) was used to determine whether the occurrence of DMs was related to the high genomic instability in human carcinoma cells. We analyzed SCE frequencies in two groups of cell lines: the first group contained DM-positive cell lines such as UACC-1598, SK-PN-DW, and NCI-N87 carcinomas, while the second group comprised DM-negative cell lines including HO-8910, U251, and MGC-803. RESULTS: The data showed that SCE was significantly increased in the DM-positive cells as compared to the DM-negative cells. In addition, there was a positive correlation between the incidence of DMs and the SCE frequency in the UACC-1598, SK-PN-DW, and NCI-N87 carcinoma cells. CONCLUSIONS: Because SCE can reflect general genome instability, it is suggested that the DMs are likely to be closely associated with genomic instability in carcinoma cells. Meanwhile, SCE may be involved in the malignant progression of DM-positive cancers.

Predicting chemosensitivity to gemcitabine and cisplatin based on gene polymorphisms and mRNA expression in non-small-cell lung cancer cells.

AIM: We used a panel of 17 non-small-cell lung cancer cell lines to investigate whether the presence of polymorphisms in the RRM1, ERCC1, ABCB1 and MTHFR genes and alterations in their mRNA expression can affect the in vitro chemosensitivity to cisplatin and gemcitabine. MATERIALS & METHODS: Polymorphisms in these genes were evaluated by direct sequencing. mRNA expression levels were assessed by realtime PCR. In vitro chemosensitivity to cisplatin and gemcitabine was expressed as IC50 values, using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. RESULTS: There was a significant, positive correlation between RRM1 mRNA expression and IC50 values for gemcitabine (r = 0.6533, p = 0.0045), and there was a significant, negative correlation between ABCB1 mRNA expression and IC50 values for cisplatin (r = -0.5459, p = 0.0287). When examining the association between the polymorphisms and IC50, we found that only the MTHFR 1298A>C polymorphism showed a tendency to be more chemosensitive to gemcitabine (p = 0.0440). CONCLUSION: These in vitro results suggest that mRNA expression levels of the RRM1 and ABCB1 genes may be useful indicators of chemosensitivity to gemcitabine and cisplatin, respectively. The MTHFR 1298A>C polymorphism was associated with gemcitabine chemosensitivity, which require further functional analysis with co-expressed genes and should be explored in prospective clinical studies to determine its potential clinical application as a predictive biomarker. Original submitted 11 February 2014; Revision submitted 3 November 2014.

Novel role for non-homologous end joining in the formation of double minutes in methotrexate-resistant colon cancer cells.

BACKGROUND: Gene amplification is a frequent manifestation of genomic instability that plays a role in tumour progression and development of drug resistance. It is manifested cytogenetically as extrachromosomal double minutes (DMs) or intrachromosomal homogeneously staining regions (HSRs). To better understand the molecular mechanism by which HSRs and DMs are formed and how they relate to the development of methotrexate (MTX) resistance, we used two model systems of MTX-resistant HT-29 colon cancer cell lines harbouring amplified DHFR primarily in (i) HSRs and (ii) DMs. RESULTS: In DM-containing cells, we found increased expression of non-homologous end joining (NHEJ) proteins. Depletion or inhibition of DNA-PKcs, a key NHEJ protein, caused decreased DHFR amplification, disappearance of DMs, increased formation of micronuclei or nuclear buds, which correlated with the elimination of DHFR, and increased sensitivity to MTX. These findings indicate for the first time that NHEJ plays a specific role in DM formation, and that increased MTX sensitivity of DM-containing cells depleted of DNA-PKcs results from DHFR elimination. Conversely, in HSR-containing cells, we found no significant change in the expression of NHEJ proteins. Depletion of DNA-PKcs had no effect on DHFR amplification and resulted in only a modest increase in sensitivity to MTX. Interestingly, both DM-containing and HSR-containing cells exhibited decreased proliferation upon DNA-PKcs depletion. CONCLUSIONS: We demonstrate a novel specific role for NHEJ in the formation of DMs, but not HSRs, in MTX-resistant cells, and that NHEJ may be targeted for the treatment of MTX-resistant colon cancer.

Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells.

Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, ß-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells.

Double minute chromosomes (DMs) are extrachromosomal cytogenetic structures found in tumour cells. As hallmarks of gene amplification, DMs often carry oncogenes and drug-resistance genes and play important roles in malignant tumour progression and drug resistance. The mitogen-activated protein kinase (MAPK) signalling pathway is frequently dysregulated in human malignant tumours, which induces genomic instability, but it remains unclear whether a close relationship exists between MAPK signalling and DMs. In the present study, we focused on three major components of MAPK signalling, ERK1/2, JNK1/2/3 and p38, to investigate the relationship between MAPK and DM production in tumour cells. We found that the constitutive phosphorylation of ERK1/2, but not JNK1/2/3 and p38, was closely associated with DMs in tumour cells. Inhibition of ERK1/2 activation in DM-containing and ERK1/2 constitutively phosphorylated tumour cells was able to markedly decrease the number of DMs, as well as the degree of amplification and expression of DM-carried genes. The mechanism was found to be an increasing tendency of DM DNA to break, become enveloped into micronuclei (MNs) and excluded from the tumour cells during the S/G2 phases of the cell cycle, events that accompanied the reversion of malignant behaviour. Our study reveals a linkage between ERK1/2 activation and DM stability in tumour cells.

S-allylmercaptocysteine promotes MAPK inhibitor-induced apoptosis by activating the TGF-ß signaling pathway in cancer cells.

S-allylmercaptocysteine (SAMC), one of the water-soluble organosulfur garlic derivatives, can induce the apoptosis of many types of cancer cells through the MAPK signaling pathway. The TGF-ß signaling pathway also plays a pivotal role in the process of oncogenesis, and has a certain crosstalk with the MAPK pathway. In the present study, hepatocellular carcinoma cell line HepG2 with an intact TGF-ß signal and colon cancer cell line SW620 with an imperfect TGF-ß signal were selected to ascertain whether SAMC induces the apoptosis of cancer cells by TGF-ß signaling. In both cell lines treated with MAPK inhibitors and SAMC, an increased apoptosis rate was observed by electron microscopy, TUNEL and flow cytometric assays. Immunohistochemistry and western blot assays showed that SAMC induced the apoptosis of cancer cells by activating TGF-ß1, TßRII, p-smad2/3, smad4 and smad7 signals, and promoting Bim expression while decreasing Bcl-2 expression and finally activating the mitochondrial apoptosis pathway proteins caspase-3 and caspase-9 in the HepG2 cell line. In contrast, in the SW620 cell line, the apoptosis induced by SAMC only affected TGF-ß1 and smad7 signals, and promoted the expression of Bax and Bad and finally activated the mitochondrial apoptosis pathway protein caspase-9. When we compare the apoptosis rate in both cell lines, a significantly lower apoptosis rate was noted in the SW620 cell line than the rate noted in the HepG2 cell line. In summary, SAMC induces the apoptosis of cancer cells by activating the TGF-ß signaling pathway, after MAPK signaling is inhibited.

Expulsion of micronuclei containing amplified genes contributes to a decrease in double minute chromosomes from malignant tumor cells.

Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM-carried genes remains to be clarified. The human colorectal cancer cell line NCI-H716 and human malignant primitive neuroectodermal tumor cell line SK-PN-DW are known to contain many DMs. To examine the amplification of DM-carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI-H716 and SK-PN-DW tumor cells. We identified the amplification regions and the DM-carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM-carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM-carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM-carried genes led to a decrease in the number of DMs and reduced the amplification of the DM-carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of γH2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM-carried genes resulted in a decrease in cell proliferation and invasion ability.

Activation of STAT3 in human gastric cancer cells via interleukin (IL)-6-type cytokine signaling correlates with clinical implications.

BACKGROUND: The signal transducers and activators of transcription 3 (STAT3) signaling pathway plays important roles in oncogenesis, angiogenesis, immunity, and tumor cell invasion. In the present study, we investigated the association of interleukin (IL)-6/STAT3 signaling pathway with T lymphocytes and clinical implication in patients with gastric cancer. METHODS: Seventy one patients who underwent gastrectomy due to gastric adenocarcinoma were studied. Blood samples were collected before and after surgical gastrectomy to quantify the levels of IL-6, IL-10 and VEGF using an enzyme-linked immunosorbent assay, as well as T lymphocyte subsets (CD3(+), CD4(+), CD8(+), CD4(+)/CD8(+)) and natural killer (NK) cells by a flow cytometry. Furthermore, the expression of IL-6, survivin, STAT3, STAT3 phosphorylation (p-STAT3), and VEGF were determined in human gastric cancer and adjacent normal mucosa through Western blot and immunohistochemistry. RESULTS: Postoperative levels of IL-6, IL-10 and VEGF in serum were significantly lower than preoperative levels. Percentages of T-cell subsets and NK cells in blood were significantly increased after postoperative-week 1 as compared to preoperative group, which was further augmented at 1 month after gastrectomy. In addition, the expression of IL-6, survivin, STAT3, p-STAT3, and VEGF were increased in human gastric cancer tissues as compared to adjacent normal mucosa. Their expression was associated with TNM stage of gastric cancer. The level of STAT3 activation in clinical samples was correlated with IL-6 expression. All gastric tumor samples, which expressed p-STAT3, also expressed IL-6 with weak expression detected in adjacent normal mucosa. CONCLUSION: Increased IL-6-induced activation of STAT3 was observed in neoplastic gastric tissue, which positively correlated with tumor progression. Moreover, IL-6 and STAT3 downstream signals such as IL-10 and VEGF were reduced in patients after removal of gastric cancer as compared to pre-operation. Therefore, inhibition of the IL-6/STAT3 signaling pathway may provide a new therapeutic strategy against gastric cancer.