Dichloroacetate enhances the antitumor efficacy of chemotherapeutic agents via inhibiting autophagy in non-small-cell lung cancer.

BACKGROUND: Chemotherapy is still the primary adjuvant strategy of cancer therapy; however, the emergence of multi-drug resistance has been a cause for concern. Autophagy has been demonstrated to have a protective role against chemotherapeutic drugs in cancer cells, and autophagy inhibition is generally considered to be a promising therapeutic strategy. However, the paucity of effective and specific autophagy inhibitors limits its application. PURPOSE: The objective of this study was to explore the effect of DCA, small molecular anti-tumor agent, on the autophagy regulation and chemosensitization in NSCLC cells. METHODS: We investigated the autophagy regulation of dichloroacetate (DCA) by laser confocal microscopy and western blotting in A549 and H1975 cell lines. The MTT assay and flow cytometry was performed for explore the chemosensitization effectiveness of DCA. The results were verified with subcutaneous tumor model in nude mice and the immunohistochemistry was applied for assessing the level of cell apoptosis and autophagy in vivo post treatment. RESULTS: We found that DCA, which exhibited antitumor properties in various carcinoma models, induced apoptosis of non-small cell lung cancer cells (NSCLC) by inhibiting cancer cell autophagy. Furthermore, Perifosine, an AKT inhibitor, can greatly weaken the capacity of inducing apoptosis by DCA. The results indicate that the AKT-mTOR pathway, a main negative regulator of autophagy, is involved in the DCA-induced inhibition of autophagy. Then, we detected the effectiveness of autophagy inhibition by DCA. When used in co-treatment with the chemotherapeutic drug paclitaxel (PTX), DCA markedly decreased cell autophagy, enhanced apoptosis and inhibited proliferation in A549 and H1975 cells. The results of the xenograft experiment demonstrate that co-treatment of PTX and DCA can significantly decrease cell proliferation in vivo and prolong the survival of mice. CONCLUSION: Our results suggest that DCA can inhibit cell autophagy induced by chemotherapeutics, providing a new avenue for cancer chemotherapy sensitization.

Association between Th17-related cytokines and risk of non-small cell lung cancer among patients with or without chronic obstructive pulmonary disease.

BACKGROUND: CD4 (+) T helper 17 (Th17) cells play critical roles in inflammation and tumor development. The involvement of Th17 cells in chronic obstructive pulmonary disease (COPD)-type inflammation-associated lung cancer has also been confirmed in animal models. However, to the authors' knowledge, it is unknown whether the role of Th17 cells is different in patients with lung cancer complicated with COPD compared with those without COPD. In the current study, the authors attempted to determine the association between the circulating levels of Th17-related cytokines and the clinical characteristics of non-small cell lung cancer (NSCLC) in patients with or without COPD. METHODS: The authors designed a matched case-control study that included 70 patients with NSCLC with COPD, 148 patients with NSCLC without COPD, and 148 healthy controls. The data regarding the clinicopathological features of these participants were collected. Circulating levels of Th17-related cytokines, including interleukin (IL) 23 (IL-23), IL-17A, IL-17F, IL-22, and tumor necrosis factor-α, were measured. RESULTS: The circulating levels of IL-23, IL-17A, IL-17F, IL-22, and tumor necrosis factor-α were found to be significantly higher in the patients with NSCLC compared with the healthy controls (P<.05). The elevated levels were found to be significantly associated with lung cancer risk (P<.05). However, no significant differences were found between patients with NSCLC with COPD and patients without COPD. It is interesting to note that, among patients with NSCLC without COPD, the levels of these cytokines were consistently higher among patients with stage I to stage IIIA disease compared with those with stage IIIB to stage IV disease (P<.05). In addition, the 5 Th17-related cytokines demonstrated pairwise correlations, with Spearman rank correlation coefficients of 0.646 to 0.888 (P<.05). CONCLUSIONS: The results of the current study indicate a clear association between the Th17-related cytokine profile and the risk of NSCLC complicated by the presence or absence of COPD.

Hypoxia-induced SOCS3 is limiting STAT3 phosphorylation and NF-κB activation in congenital heart disease.

Suppressor of cytokine signaling 3 (SOCS3) is a critical attenuator of the JAK-STAT signaling pathway, and it is involved in mediating the intensity and duration of STAT3 activation in the process of myocardial protection. Nuclear factor-κB (NF-κB) has emerged as a decisive transcription factor in cardiac myocyte compensatory responses to stress that enhance survival. However, the expression, activation and regulation of this signaling molecule in response to hypoxic stress have not been elucidated. We investigated 40 infants with cyanotic or acyanotic cardiac defects, as well as H9c2 embryonic rat cardiomyocytes, to examine the effect of hypoxia on the expression or activation of SOCS3, STAT3 and NF-κB in vivo and in vitro. We found an increase in endogenous cardiac SOCS3, p-STAT3 and AC-RelA activation in the myocardium of infants with cyanotic cardiac defects. In hypoxic cultivated H9c2 cells, SOCS3, STAT3 and AC-RelA activity slowly increased and then reached a stable expression. We evaluated the interaction of SOCS3 with STAT3 and NF-κB by transfecting the SOCS3 plasmid to hypoxic cultured H9c2 cells. Forced expression of SOCS3suppressed tyrosine phosphorylation of STAT3 and transcription of the C-myc and interleukin-6 genes. AC-RelA activation was also suppressed by over expression of SOCS3. These findings suggest that the mechanism of a positive transactivation loop that maintains higher levels of NF-κB and p-STAT3 and the negative feedback factor SOCS3, which maintains balanced NF-κB and p-STAT3 activities, is important in the process of myocardial adaptation to chronic hypoxia. SOCS3 is a rapid hypoxia inducible gene and acts to inhibit activation of the cellular signaling pathway in a classical negative feedback loop. Upregulated SOCS3 might play an important role in cardiocytes during chronic hypoxia as SOCS3 regulates cell signaling crosstalking between NF-κB and p-STAT3 under stressful conditions.

Restoration of SOCS3 suppresses human lung adenocarcinoma cell growth by downregulating activation of Erk1/2, Akt apart from STAT3.

SOCS3 is regarded as a major negative regulator of STAT3. Recent evidence indicates that SOCS3 regulates strength and duration of other signaling pathways including ras/ERK1/2/MAPK, PI3-K/Akt in non-malignant cells. The repression or silence of SOCS3 expression in a few tumor types has led to speculation that loss of SOCS3 gene is closely related to deregulation of multiple signal pathways during tumorigenesis. However, apart from STAT3, little is known in malignant cells about the mechanism by which SOCS3 modulates other intracellular signal cascades such as Erk1/2 and Akt, whose aberrant activation has been implicated in many human tumors. Expression of SOCS3 proved deficient in human lung adenocarcinoma A549 cells, and forced expression of SOCS3 resulted in growth inhibition. Growth suppression due to SOCS3 was associated with attenuated activation of Erk1/2, Akt as well as STAT3. The results suggested that SOCS3, as negative regulators of cytokine signaling, might maintain homeostasis by regulating multiple signaling pathways and reverse cell malignant behavior.

[Effects of cytokine signaling 3 suppressors gene on c-fos and c-jun mRNA expression and proliferation of pulmonary arterial smooth muscle cells in rat under hypoxia].

OBJECTIVE: To explore the effects of suppressors of cytokine signaling (SOCS)3 gene on expression of c-fos, c-jun mRNA and proliferation of rat pulmonary arterial smooth muscle cells(PASMCs) under hypoxia. METHODS: PASMCs were co-transfected with pEFSOCS3 and pSV2neo by liposome, and then expression of SOCS3 protein was detected by immunocytochemistry. After PASMCs were exposed to normoxic and hypoxia at various time points respectively, expression of c-fos and c-jun mRNA was assessed by semi-quantitive RT-PCR. Flow cytometric DNA analysis was used to detect cell cycles. RESULTS: Expression of SOCS3 protein was confirmed by Western blot in PASMCs transfected with SOCS3 gene. The c-fos mRNA level in control cells peaked at 2 h of hypoxia and declined at 4 h, then peaked at 8 h secondly and declined at 12 h. C-fos mRNA level in SOCS3 gene-transfected cells at 2 h and 8 h exposed to hypoxia was lower than that in control cells at the same time points respectively (P < 0.01). The c-jun mRNA level increased at 2 h after exposure of control cells to hypoxia, peaked at 6 h of hypoxia and declined to the basal levels at 12 h. C-jun mRNA level of SOCS3 gene-transfected cells at 2 h, 4 h, 6 h, 8 h under hypoxia was lower than that in controls cells at the same time points. Compared with control PASMCs, cells in transfected with SOCS3 gene at G(1)/G(0) phase increased and those at S + G(2)/M phase decreased under normoxic and hypoxia (P < 0.01). CONCLUSION: Hypoxia induced expression of c-fos and c-jun genes, which might play an vital role in the early stage of PASMCs proliferation. SOCS3 protein may inhibit proliferation of PASMCs by lowering the tyrosine-phosphorylated level of STAT3 protein under hypoxia.

[Effects of transfected SOCS3 gene on proliferation of human lung adenocarcinoma cell line A549].

BACKGROUND & OBJECTIVE: Previous studies showed that signal transducers and activators of transcription 3(STAT3) could be constantly activated in many kinds of tumor cells; constantly activated STAT3 could make cell malignant transformation, and enhance the malignant proliferation of tumor cells. The suppressors of cytokine signaling (SOCS) proteins, a recently identified family, could suppress the tyrosine-phosphorylated process of STAT protein to negatively regulate STAT-mediated cytokine signaling, which is closely related to cell survival, proliferation, and apoptosis. Among the SOCS protein family, SOCS3 is the most potent inhibitors of cytokine-induced signaling. This experiment was to investigate the effect of SOCS3 on proliferation of human lung adenocarcinoma cell line A549. METHODS: A549 cells were co-transfected with pEFSOCS3 and pSV2neo by liposome, expression of SOCS3 mRNA and protein was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and immunocytochemistry. Tyrosine-phosphorylated STAT3 level was measured by Western blot analysis. MTT assay and (3)-TdR incorporation were used to measure the cell proliferation in pEFSOCS3-transfected group, and control group (non-transfected, and pEF-transfected A549). RESULTS: Compared with control group, tyrosine-phosphorylated STAT3 level suppressed greatly in pEFSOCS3-transfected A549 cells (P< 0.01). The inhibitory rate of cell growth in pEFSOCS3-transfected group was 40.58%, and in control group was 32.23%. (3)-TdR incorporation (48 056+/-1 331) in pEFSOCS3-transfected group was significantly lower than that of control group (81,481+/-2,584, and 75,590+/-2,678, P< 0.01). CONCLUSION: SOCS3 protein might inhibit proliferation of A549 cells by lowering the tyrosine-phosphorylated level of STAT3 protein.