Cunzhong Yuan

Increased Frequencies of Th22 Cells as well as Th17 Cells in the Peripheral Blood of Patients with Ankylosing Spondylitis and Rheumatoid Arthritis

Background T-helper (Th) 22 is involved in the pathogenesis of inflammatory diseases. The roles of Th22 cells in the pathophysiological of ankylosing spondylitis (AS) and rheumatoid arthritis (RA) remain unsettled. So we examined the frequencies of Th22 cells, Th17 cells and Th1 cells in peripheral blood (PB) from patients with AS and patients with RA compared with both healthy controls as well as patients with osteoarthritis. Design and Methods We studied 32 AS patients, 20 RA patients, 10 OA patients and 20 healthy controls. The expression of IL-22, IL-17 and IFN-γ were examined in AS, RA, OA patients and healthy controls by flow cytometry. Plasma IL-22 and IL-17 levels were examined by enzyme-linked immunosorbent assay. Results Th22 cells, Th17 cells and interleukin-22 were significantly elevated in AS and RA patients compared with OA patients and healthy controls. Moreover, Th22 cells showed positive correlation with Th17 cells as well as interleukin-22 in AS and RA patients. However, positive correlation between IL-22 and Th17 cells was only found in AS patients not in RA patients. In addition, the percentages of both Th22 cells and Th17 cells correlated positively with disease activity only in RA patients not in AS patients. Conclusions The frequencies of both Th22 cells and Th17 cells were elevated in PB from patients with AS and patients with RA. These findings suggest that Th22 cells and Th17 cells may be implicated in the pathogenesis of AS and RA, and Th22 cells and Th17 cells may be reasonable cellular targets for therapeutic intervention.

Huaier aqueous extract inhibits proliferation of breast cancer cells by inducing apoptosis

Aqueous extract of Trametes robiniophila murr (Huaier) has been commonly used in China for cancer complementary therapy in recent years; however, the mechanisms of its anticancer effects are largely unknown. In the present study, we aim to investigate its inhibitory effect on both MCF‐7 and MDA‐MB‐231 cells, and explore the possible mechanisms of its anticancer effect. Cell viability and motility were measured by MTT and invasive assays, migration and scratch assays in vitro, respectively. The distribution of cell cycle, PI‐Annexin‐V staining and Rhodamine 123 assay were analyzed by flow cytometry, and western blot were used to test the apoptotic pathways. We found that Huaier extract could strongly inhibit cell viability of MCF‐7 and MDA‐MB‐231 cells in a time‐ and dose‐dependent manner; however, MDA‐MB‐231 cells showed more susceptibility to the treatment. Furthermore, cell invasiveness and migration were also suppressed with exposure to Huaier extract. We also indicated that Huaier could induce G0/G1 cell‐cycle arrest, p53 accumulation and activation selectively in MCF‐7 cells. Inspiringly, the PI‐Annexin‐V staining assay and western blot analysis confirmed cell apoptosis executed by caspase‐3. Decreased mitochondrial membrane potential by Rhodamine 123 assay and down‐regulation of Bcl‐2 and up‐regulation of BCL2‐associated X protein (BAX) indicated that Huaier induced apoptosis through the mitochondrial pathway. Caspase activation during Huaier‐induced apoptosis was confirmed by pan‐caspase inhibitor, Z‐VAD‐fmk. As expected, the inhibitor decreased Huaier‐induced apoptosis in both cell lines. Based on our findings, Huaier can induce cell apoptosis in both ER‐positive and ER‐negative breast cancer cell lines and is an effective complementary agent for breast cancer treatment. (Cancer Sci 2010; 101: 2375–2383)

Metadherin enhances the invasiveness of breast cancer cells by inducing epithelial to mesenchymal transition

The epithelial–mesenchymal transition (EMT) is a process in which polarized epithelial cells are converted into motile mesenchymal cells. During cancer development, EMT is conducive to tumor dissemination and metastatic spread. While overexpression of metadherin (MTDH) in breast cancer cell lines and tissues has been found to be associated with aggressive tumor behavior, its precise role in invasion and metastasis is largely unknown. Here we report that MTDH overexpression could significantly enhance the invasion and migration of breast cancer cells by inducing EMT. Metadherin overexpression led to upregulation of mesenchymal marker fibronectin, downregulation of epithelial marker E‐cadherin, and the nuclear accumulation of beta‐catenin. Also, transcription factors Snail and Slug were upregulated in breast cancer cells overexpressing MTDH. Overexpression of MTDH enhanced the invasiveness and migration ability of breast cancer cells in vitro. In addition, overexpression of MTDH led to increased acquisition of CD44+/CD24−/low markers that are characteristic of breast cancer stem cells. We also showed that NF‐kappa was involved in the expression of EMT‐related markers. Taken together, our results suggest that MTDH could promote EMT in breast cancer cells in driving the progression of their aggressive behavior. (Cancer Sci 2011; 102: 1151–1157)

Metadherin Mediates Lipopolysaccharide-Induced Migration and Invasion of Breast Cancer Cells

Background Breast cancer is the most prevalent cancer in women worldwide and metastatic breast cancer has very poor prognosis. Inflammation has been implicated in migration and metastasis of breast cancer, although the exact molecular mechanism remains elusive. Principal Findings We show that the pro-inflammatory endotoxin Lipopolysaccharide (LPS) upregulates the expression of Metadherin (MTDH), a recently identified oncogene, in a number of breast cancer lines. Stable knockdown of MTDH by shRNA in human breast MDA-MB-231 cells abolishes LPS-induced cell migration and invasion as determined by several in vitro assays. In addition, knockdown of MTDH diminishes Nuclear Factor-kappa B (NF-κB) activation by LPS and inhibited LPS-induced IL-8 and MMP-9 production. Conclusions These results strongly suggest that MTDH is a pivotal molecule in inflammation-mediated tumor metastasis. Since NF-κB, IL-8 and MMP-9 play roles in LPS-induced invasion or metastasis, the mechanism of MTDH-promoted invasion and metastasis may be through the activation of NF-κB, IL-8 and MMP-9, also suggesting a role of MTDH in regulating both inflammatory responses and inflammation-associated tumor invasion. These findings indicate that MTDH is involved in inflammation-induced tumor progression, and support that MTDH targeting therapy may hold promising prospects in treating breast cancer.

Identification of Novel Variants of Metadherin in Breast Cancer

Metadherin (MTDH, also known as AEG-1, and Lyric) has been demonstrated to play a potential role in several significant aspects of tumor progression. It has been reported that overexpression of MTDH is associated with progression of disease and poorer prognosis in breast cancer. However, there are no studies to date assessing variants of the MTDH gene and their potential relationship with breast cancer susceptibility. Thus, we investigated all variants of the MTDH gene and explored the association of the variants with breast cancer development. Our cohort consisted of full-length gene sequencing of 108 breast cancer cases and 100 healthy controls; variants were detected in 11 breast cancer cases and 13 controls. Among the variants detected, 9 novel variants were discovered and 2 were found to be associated with the susceptibility of breast cancer. However, additional studies need to be conducted in larger sample sizes to validate these findings and to further investigate whether these variants are prognostic in breast cancer patients.

Low-dose radiation-induced epithelial-mesenchymal transition through NF-κB in cervical cancer cells

Cervical cancer is the leading cause of death from cancer among women. Radiotherapy for cervical cancer is an effective treatment method; however, the response to radiotherapy varies among patients. Epithelial-mesenchymal transition (EMT) is a morphogenesis process involved in embryonic and organismal development. During tumour progression, EMT may enhance cancer cell invasion, promoting tumour metastasis. We hypothesised that EMT was involved in the enhanced invasiveness of cervical cancer cells after low-dose radiation and aimed to elucidate the underlying mechanism of this process in low-dose radiation of cervical cancer. The irradiated cells (FIR cells) were derived from the parental cells (N cells) with a cumulative dose of 75 Gy. After resting and reorganisation, the effect of low-dose radiation on the FIR cells was analysed. The expression of E-cadherin, N-cadherin and p65 was detected by real-time qPCR and western blotting in parental cancer cells and irradiated cancer cells. Motility was detected using the migration/invasion assay. After silencing of NF-κB p65 expression using siRNA against p65, the expression of E-cadherin and N-cadherin was examined by real‑time qPCR and western blotting. We found that low-dose radiation induced morphological changes of cells. The expression of epithelial markers was downregulated and mesenchymal markers were induced in irradiated cells, both of which are characteristics of EMT. Additionally, in irradiated cells, migration and invasion were enhanced and the expression of p65 was increased. To investigate whether p65 was involved in EMT, we silenced the expression of p65 in irradiated cells using siRNA and found that the features of EMT were suppressed. In summary, p65-regulated EMT induced by low-dose irradiation of cervical cancer cell lines promoted the invasiveness and metastasis of cervical cancer cells. The reversal of EMT may be a new therapeutic target for improving the effectiveness of radiotherapy for cervical cancer.

BCL-2 (-938C > A) polymorphism is associated with breast cancer susceptibility

BackgroundBCL-2 (B-cell leukemia/lymphoma 2) gene has been demonstrated to be associated with breast cancer development and a single nucleotide polymorphism (SNP; -938C > A) has been identified recently. To investigate whether this polymorphism functions as a modifier of breast cancer development, we analyzed the distribution of genotype frequency, as well as the association of genotype with clinicopathological characteristics. Furthermore, we also studied the effects of this SNP on Bcl-2 expression in vitro.MethodsWe genotyped the BCL-2 (-938C > A) in 114 patients and 107 controls, and analyzed the estrogen receptor (ER), progestogen receptor (PR), C-erbB2 and Ki67 status with immunohistochemistry (IHC). Different Bcl-2 protein levels in breast cancer cell lines were determined using western blot. Logistic regression model was applied in statistical analysis.ResultsWe found that homozygous AA genotype was associated with an increased risk (AA vs AC+CC) by 2.37-fold for breast cancer development and significant association was observed between nodal status and different genotypes of BCL-2 (-938C > A) (p = 0.014). AA genotype was more likely to develop into lobular breast cancer (p = 0.036). The result of western blot analysis indicated that allele A was associated with the lower level of Bcl-2 expression in breast cancer cell lines.ConclusionsAA genotype of BCL-2 (-938C > A) is associated with susceptibility of breast cancer, and this genotype is only associated with the nodal status and pathological diagnosis of breast cancer. The polymorphism has an effect on Bcl-2 expression but needs further investigation.

Inactivation of Notch signaling reverses the Th17/Treg imbalance in cells from patients with immune thrombocytopenia.

T helper 17 (Th17) cells and regulatory T (Treg) cells, along with Th1 and Th2 cells, may contribute to the development of immune thrombocytopenia (ITP). The imbalance of Th17/Treg toward Th17 cells has been shown to play a pivotal role in the peripheral immune response. Notch signaling has been implicated in peripheral T-cell activation and effector cell differentiation. However, the role of Th17/Treg in the pathogenesis of ITP and the effect of Notch signaling on Th17/Treg imbalances remain largely elusive in ITP. In vitro, we treated peripheral blood mononuclear cells (PBMCs) from ITP and healthy controls with γ-secretase inhibitor (DAPT). Th17 cells and Treg cells were measured by flow cytometry and IL-17, IL-21, and IL-10 secretion by enzyme immunoassay technique. The mRNA expression of Ntoch1, Hes1, Hey1, RORγt, and Foxp3 was investigated by RT-PCR. Cell proliferation and apoptosis were determined by the Cell Counting Kit-8 and apoptosis detection kit. We demonstrated that DAPT was effective in inhibiting mRNA expression of Notch signaling molecules. In untreated cultured PBMCs from ITP patients, we observed elevated Th17 cell and IL-21 levels and RORγt mRNA expression, decreased Treg cells and Foxp3 mRNA expression, and an increased ratio of Th17/Treg and RORγt/Foxp3. After inactivating Notch signal by DAPT, Th17 cells and Th17/Treg ratio were dose dependently decreased and accompanied by the reduction of IL-17 in culture supernatants and RORγt mRNA expression in ITP patients. However, no significant difference was found for Treg cells and Foxp3 mRNA expression, RORγt/Foxp3 ratio, and IL-21 and IL-10 levels after DAPT treatment in ITP patients. We also present evidence that the effect of DAPT inhibition on the Th17 cell response was associated with downregulation of RORγt and IL-17 transcription using human in vitro polarization. In conclusion, our findings highlight the importance of Notch signaling in Th17/Treg imbalances in ITP. Inactivation of Notch signaling might be a potential immunoregulatory strategy in ITP patients.

The MTDH (−470G>A) Polymorphism Is Associated with Ovarian Cancer Susceptibility

MTDH(metadherin), an important oncogene that is widely overexpressed in various cancers, is a potential biomarker of tumor malignancy. Variants in MTDH have been associated with susceptibility to breast cancer. However, no studies assessing MTDH gene polymorphisms and their potential relationship to ovarian cancer susceptibility have been reported. Thus, we investigated the association of MTDH (−470G>A) polymorphism with ovarian cancer development in 145 ovarian cancer patients and 254 matched control subjects, using sequence analysis. We found that the MTDH (−470G>A) polymorphism was statistically correlated with ovarian cancer risk (under the additive genetic model, GG vs. GA vs AA, P = 0.042). Compared with genotypes containing the G allele (GG and GA), the AA genotype may decrease the risk of ovarian cancer (P = 0.0198, OR = 0.33, 95% CI [0.12∼0.78]). Compared with the G allele, the A allele is protective against ovarian cancer risk (P = 0.01756, OR = 0.66, 95% CI [0.46∼0.93]). Furthermore, a statistically significant association between the GG and GA+AA genotypes and the clinical stage was observed (P = 0.038). These data suggest that MTDH (−470G>A) could be a useful molecular marker for assessing ovarian cancer risk and for predicting ovarian cancer patient prognosis.

Metadherin regulates radioresistance in cervical cancer cells.

Metadherin (MTDH) promotes cancer metastasis, chemoresistance, invasion and angiogenesis. Upregulation of MTDH is correlated with both progression and poor clinical outcome of many types of cancers; however, there is currently no information regarding the role of MTDH in radiation sensitivity. Here, we investigated the effects of MTDH on the radiosensitivity of cervical cancer cells using the SiHa cell line. We discovered that cervical cancer cells in which MTDH was knocked down had significantly increased radiosensitivity as measured by a clonogenic assay. MTDH knockdown cells also had increased apoptosis and a decreased proportion of cells arrested in the G2 phase after radiation treatment. MTDH knockdown also weakened the repair of DNA double-strand breaks (DSBs) induced by radiation. These results indicate that MTDH affects the radiosensitivity of cervical cancer cells and that MTDH may be a novel target to improve cervical cancer radiation response.