Thiol oxidative stress-dependent degradation of transglutaminase2 via protein S-glutathionylation sensitizes 5-fluorouracil therapy in 5-fluorouracil-resistant colorectal cancer cells.

5-Fluorouracil (5-Fu) is a first-line drug for colorectal cancer (CRC) therapy. However, the development of 5-Fu resistance limits its chemotherapeutic effectiveness and often leads to poor prognoses of CRC. Transglutaminase 2 (TGM2), a member of the transglutaminase family, is considered to be associated with chemoresistance through apoptotic prevention in various cancers including CRC. TGM2 was found to be overexpressed in two 5-Fu-resistant CRC cell lines and down-regulated by increased thiol oxidative stress induced by inhibition of glutathione reductase (GR). The present study aimed to explore the role of TGM2 in 5-Fu-resistant CRC and the mechanism of action by which the elevated thiol oxidative stress down-regulates TGM2 protein level. The results revealed that 5-Fu-resistance induced by overexpression of TGM2 in CRC cells was reversed through up-regulation of thiol oxidative stress. Knockdown of TGM2 increased the chemosensitivity of CRC cells to 5-Fu. Thiol oxidative stress potentially enhanced the therapeutic effect of 5-Fu in the resistant CRC cells by promotion of 5-Fu-induced apoptosis through down-regulation of TGM2. The elevated thiol oxidative stress increased the S-glutathionylation of TGM2 and led to proteasomal degradation of TGM2. Furthermore, Cys193 was identified as the S-glutathionylation site in TGM2, and its mutation resulted in thiol oxidative stress-mediated CRC cell apoptotic resistance. TGM2-induced EMT was also suppressed by the elevated thiol oxidative stress. A xenograft tumor model confirmed the effect of thiol oxidative stress in the reversal of 5-Fu resistance in CRC cells in vivo. TGM2 protein expression level was found to be significantly higher in human CRC specimens than in non-cancerous colorectal tissues. Taken together, the present data suggest an important role of TGM2 in 5-Fu resistance in CRC cells. Up-regulation of thiol oxidative stress could be a potential therapeutic approach for treating 5-Fu-resistant CRC and TGM2 may serve as a potential therapeutic target of thiol oxidative stress.

Establishment and Characterization of a CD20-Positive NK/T-Cell Lymphoma Cell Line.

BACKGROUND: CD20 positive NK/T-cell lymphoma is extremely rare and difficult for clinical treatment. Due to the lack of an established cell model for this disease, less is known about its biological characterization and potential therapeutic options. METHODS: A cell line of NK/T-cell lymphoma, which was enriched by magnetic sorting with proper cell surface markers (CD56) from peripheral blood mononuclear cells (PBMCs) drawn from a 21-year-old male patient with nasal angiocentric NK/T-cell lymphoma, was designated as ZQNK-29. Immunophenotypic analysis of ZQNK-29 was performed by flow cytometric and immunohistochemical analysis. Comparative genomic hybridization (CGH) analysis was used for cytogenetic analysis of ZQNK-29. Potential rearrangements of the immunoglobulin gene and Epstein-Barr virus (EBV) infection were examined by PCR and RT-PCR, respectively. RESULTS: ZQNK-29 cells express the phenotypic T-cell marker (CD3), T cell activation markers (HLA-DR), markers for both NK and cytotoxic T lymphocytes (TIA-1), and B-lineage marker CD20; however, expression of CD56 was not detected in expanded ZQNK-29 cells although this NK cell surface marker was used as one of selective cell surface markers for the initial isolation of NK/T cells. RT-PCR analysis showed that the pattern of gene expressions for infected EBV was latency type III, with the expressions of LMP1, EBNA-1, and EBNA-2; no rearrangements were found in the heavy-chain of the immunoglobulin gene or in the y chain of the T cell receptors (TCRs) gene. CGH analysis demonstrated that ZQNK-29 possessed an abnormal karyotype, 46XY, 1p (dist)+, 4p (dist)+, 4q (mid)-, 5q (mid)-, 9q (dist)+, 16p (dist)+, 16q (dist)+, 17p+, 17q (dist)+, 19q (dist)+, 20p+, 20q+, 21q+, and 22q+. Of these, 1p (dist)+, which has been confirmed to be mitochondrial DNA amplification, is believed to be mainly caused by EBV infection. CONCLUSIONS: ZQNK-29 is a well characterized premature human NK/T-cell lymphoma cell line with expression of the B-cell marker CD20 and will provide a useful pre-clinic model for characterization and potential therapeutic studies of the aggressive NK/T-cell lymphoma.

Clinicopathological and prognostic significance of serum cytokine levels in breast cancer.

BACKGROUND: This study was designed to examine the serum levels of six cytokines (IFN-gamma, TNF-alpha, IL-2, IL-4, IL-5, and IL-10) in treated and untreated breast cancer patients and assess their clinical significance. The correlation of the Th1/Th2 type cytokine levels and the clinicopathologic variables was further evaluated. METHODS: Cytometric Bead Array (CBA) was used to examine the levels of Th1 cytokines (TNF-alpha, IFN-gamma, and IL-2) and Th2 cytokines (IL-4, IL-5, and IL-10) in serum of 36 untreated and 73 treated breast cancer patients and 51 healthy females as control. RESULTS: In the untreated group, the levels of IFN-gamma, IL-4 and IL-5 were significantly higher than in control group (p < 0.05). IFN-gamma, IL-2, IL-5, and IL-10 levels were higher in treated group than in untreated group (p < 0.05); IFN-gamma/IL-4 ratio of the treated group was higher than the untreated group (p < 0.05). Meanwhile, the cytokine levels were significantly different in different pTNM stages and lymph node involvement groups. Survival analysis revealed that the IFN-gamma/IL-4 ratio, pTNM stage, and lymph node involvement affected the survival of breast cancer patients. The pTNM stage was an independent significant prognostic factor. CONCLUSIONS: Breast cancer patients presented a Th1/Th2 imbalance and immune function disorder, which correlated with pTNM stage and lymph node involvement. Higher IFN-gamma/IL-4 ratio predicted a favorable outcome in breast cancer patients.

A pan-cancer analysis of SLC1A5 in human cancers.

Background: The alanine-serine-cysteine transporter 2, ASCT2 (solute carrier family 1 member 5, SLC1A5), is a major transporter of the amino acid, glutamine. Although SLC1A5 has been reported to be associated with some types of cancer, less pan-cancer analysis, which would give a comprehensive understanding of SLC1A5 across human cancers, has been carried out. Methods: We used the TCGA and GEO databases to investigate the oncogenic role of SLC1A5. We examined gene and protein expression, survival, genetic mutations, protein phosphorylation, immunocyte infiltration and the related genes correlated pathways. In HCT116 cells, SLC1A5 was silenced by siRNAs and the mRNA and protein was checked by Q-PCR and WB, respectively and the cellular function was assessed by CCK8, cell cycle and apoptosis. Results: We found that SLC1A5 was over-expressed in multiple types of cancer and that elevated expression of SLC1A5 was associated with poor survival in many cancers. The missense mutation of R330 H/C was associated with poor survival, especially in uterine carcinosarcoma. Furthermore, we found enhanced phosphorylation of S503 in uterine corpus endometrial carcinoma and lung adenocarcinoma. In addition, elevated SLC1A5 expression was associated with immune cell infiltration in many cancers. KEGG and GO analysis showed that SLC1A5 and its related genes were involved in central carbon metabolism in cancer, due to their amino acid transport activity. The cellular function indicated that SLC1A5 may influence the cell proliferation by affecting DNA synthesis. Conclusions: Our findings highlighted the important role of SLC1A5 in tumorigenesis and provided insights into potential cancer treatment strategies.

N­acetyl-S-(p-chlorophenylcarbamoyl)cysteine induces mitochondrial-mediated apoptosis and suppresses migration in melanoma cells.

We previously reported that N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine (NACC) induces apoptosis in human melanoma UACC-62 cells. In the present study, the molecular mechanism of NACC­induced apoptosis in melanoma cells was investigated. Briefly, the apoptosis triggered by NACC was confirmed in UACC­62 cells for shorter treatment periods. Increased activities of caspase­3 and caspase­9 but not caspase­8 were observed in the cell lysates. Western blotting showed that the pro­apoptotic protein Bax was upregulated and the anti­apoptotic protein Mcl­1 was downregulated and cytochrome c (Cyto c) was released into the cytosol. Flow cytometric analysis demonstrated that NACC induced significant mitochondrial membrane potential disruption. Significant increases in the generation of reactive oxygen species (ROS) and cytosolic calcium elevation were also observed. However, opening of the mitochondrial permeability transition pore which could be involved in Cyto c leakage from mitochondria was found to be unaffected by NACC. Taken together, all the results presented in this study including apoptotic induction, activation of the caspase­3 and ­9 cascade, upregulation of Bax, downregulation of Mcl­1, Cyto c release from the mitochondria, mitochondrial membrane potential depletion, ROS production and cytosolic calcium elevation demonstrated that NACC triggered apoptosis in the UACC­62 cells via the mitochondrial­dependent pathway. Melanoma is well­known as an aggressive and highly metastatic disease. In this study, we also investigated the effects of NACC on the migration of UACC­62 cells using the xCELLigence system. The results revealed that in vitro NACC is capable of inhibiting the migration of melanoma cells.

[Establishment of human multidrug-resistant lung carcinoma cell line (D6/MVP)].

OBJECTIVE: To establish human multidrug-resistant lung carcinoma cell line (D6/MVP) with its characteristics studied. METHODS: Intermittent administration of high-dose MMC, VDS and DDP (MVP) was used to induce human lung carcinoma cell line (D6) to a multidrug-resistant variety (D6/MVP). MTT assay was used to study the multidrug resistance of D6/MVP to multianticarcinogen. Flow cytometry was used to study the cell cycle distribution and the expression of P-gp, multidrug resistance-associated protein (MRP) and GSH/GST. RESULTS: 1. D6/MVP was resistant to many anti-tumor agents, with the IC(50) 13.3 times higher and the drug resistance 2 - 6 times higher than D6, 2. The multiplication time of D6/MVP was prolonged and the cell number of S-phase decreased while that of G1- and G(2)-phase increased and 3. The expression of P-gp and MRP was enhanced significantly (96.2% vs 51.7%), but the expression of GSH/GST kept stable. CONCLUSION: D6/MVP is a multidrug-resistant cell line possessing the basic characteristics of drug-resistance.

Unfavorable clinical implications of circulating CD44+ lymphocytes in patients with nasopharyngeal carcinoma undergoing radiochemotherapy.

BACKGROUND: To evaluate the use of cellular immunity parameters as predictors of therapy response. METHODS: Circulating lymphocytes were measued by flow cytometry in 94 nasopharyngeal carcinoma (NPC) patients following radiochemotherapy. RESULTS: Significantly decreased percentage of CD3(+), CD4(+), and CD8(+) lymphocytes, significantly increased proportion of CD44(+), CD25(+), NK lymphocytes, and an increased CD4(+)/CD8(+) ratio were indicated in NPC patients as compared with healthy controls. Circulating CD44(+) lymphocytes in both the N2/N3 and III/IV groups were significantly increased as compared to the N0/N1 and I/II groups, respectively (P<0.05). A significant decrease in CD19(+) lymphocytes was observed in the III/IV group as compared with the I/II group (P<0.05). After radiochemotherapy, NPC patients had significantly (P<0.05) decreased percentages of CD4(+), CD44(+), and CD19(+) lymphocytes and a decreased CD4(+)/CD8(+) ratio, whereas the mean percentages of CD8(+) and NK lymphocytes were significantly (P<0.05) increased. However, compared with the pre-radiochemotherapy values, no significant (P>0.05) changes in CD3(+) or CD25(+) lymphocytes were observed in the NPC-treated group. Follow-up analysis indicated significantly lower DFS for patients with high CD44(+) lymphocytes compared to those with low CD44(+) lymphocytes after radiochemotherapy. CONCLUSION: Circulating CD44(+) lymphocytes seems to be a promising criterion to predict survival in NPC patients undergoing radiochemotherapy.